CN111647529B - 4-BA paraincidentally Mycobacterium strain with rapid growth and high transformation capacity and preparation method thereof - Google Patents

Abstract

The invention relates to the field of strain breeding, in particular to a 4-BA paraincidentally mycobacterium strain with rapid growth and high transformation capacity and a preparation method thereof. The 4-BA paraincidentally Mycobacterium strain (Mycobacterium parafortuitum) with rapid growth and high transformation capacity has a classification name Mycobacterium parafortuitum BK047, a preservation unit of China center for type culture collection, and a preservation number of CCTCC M2020076. The 4-BA paraincidentally Mycobacterium strain with the rapid growth and high transformation capacity provided by the invention has the high transformation capacity and the rapid growth capacity, is beneficial to improving the sterol feeding concentration, accelerating the transformation time, reducing the production cost and improving the production efficiency.

Description

4-BA paraincidentally Mycobacterium strain with rapid growth and high transformation capacity and preparation method thereof

Technical Field

The invention relates to the field of strain breeding, in particular to a 4-BA paraincidentally mycobacterium strain with rapid growth and high transformation capacity and a preparation method thereof.

Background

Steroid drugs are a clinically indispensable drug class, and play a very important role in regulating the organism. For example, adrenocortical hormone has the functions of anti-inflammation, anti-allergy, anti-shock, anti-allergic reaction and the like, is widely used for treating skin diseases such as rheumatoid arthritis, bronchial asthma, eczema and the like, can treat and relieve refractory or dangerous diseases such as collagenous diseases, anaphylactic shock and the like, and is also an indispensable medicament for treating endocrine diseases such as Edison and the like.

The steroid is the world second largest class of drugs with sales rates inferior to antibiotics, and different molecular structures of steroid are derived from steroid intermediates. 20 alpha-hydroxymethyl-pregn-4-en-3-one (Bisnoralcoho) is called 4-BA for short, is a key steroid drug intermediate and plays an important role in steroid industrial production. The 4-BA is used as a precursor to synthesize various important steroid bulk drugs such as progesterone, 17 alpha-hydroxy progesterone, hydrocortisone, betamethasone, dexamethasone, cortisone, flumetesone, eplerenone and the like, and has important commercial value.

At present, the microbial method is mainly used for converting the phytosterol to produce 4-BA. But the problems in production are: the existing 4-BA strain has stronger transformation capability, but has longer seed growth period, thereby severely restricting the improvement of the yield, increasing the production cost and affecting the production efficiency.

Disclosure of Invention

Therefore, the invention aims to overcome the defects of the prior art that the seed growth period of the 4-BA strain is longer, the production efficiency is lower and the production cost is higher, thereby providing the 4-BA paraincidentally Mycobacterium strain with rapid growth and high transformation capacity and the preparation method thereof.

In order to solve the technical problems, the invention adopts the following technical scheme:

A4-BA paraincidentally Mycobacterium strain (Mycobacterium parafortuitum) with rapid growth and high transformation capability is named Mycobacterium parafortuitum BK047 by classification, the preservation unit is China center for type culture collection, and the preservation number is CCTCC M2020076.

A method for preparing a 4-BA paraincidentally mycobacterial strain having a rapid growth and high transformation capacity as described above, comprising the steps of:

carrying out protoplast fusion on a 4-BA strain sp1 with high transformation capacity and a 4-BA strain sp2 with rapid growth capacity in a polyethylene glycol system to obtain a fusion seed, and screening the fusion seed to obtain the 4-BA paraaccidental mycobacterium strain with rapid growth and high transformation capacity.

Further, the polyethylene glycol is PEG4000-6000, and the volume concentration is 40% -60%.

Further, the protoplast fusion includes the steps of:

preparation of protoplasts: culturing bacterial strain sp1 and bacterial strain sp2 on a liquid complete culture medium respectively, allowing cells to grow into a logarithmic growth phase, and removing walls to obtain protoplast suspensions of the bacterial strain sp1 and the bacterial strain sp2 respectively;

Protoplast regeneration: carrying out regeneration culture on protoplast suspensions of the strain sp1 and the strain sp2 on a regeneration supplementing basic solid culture medium respectively to obtain regenerated protoplast suspensions of two parents;

protoplast fusion: and respectively taking regenerated protoplast suspensions of the strain sp1 and the strain sp2, mixing, centrifuging, discarding supernatant, adding a protoplast stabilizing solution and a polyethylene glycol solution into the precipitate, and carrying out protoplast fusion.

Further, the liquid complete medium comprises the following components:

10-15g/L of peptone, 10-15g/L of glucose, 5-10g/L of yeast powder, 5-10g/L of beef extract and 1-3g/L of sodium chloride; the pH of the liquid complete medium was 7.5.

Further, the wall removing operation uses lysozyme for wall removing, the concentration of the used lysozyme solution is 60-100 mug/ml, and the wall removing operation comprises the step of enzymolysis at 36-40 ℃ for 35-50 min.

Further, the regeneration-supplemented, substantially solid medium comprises the following components: 10-20g/L of glucose, 5-10g/L of ammonium sulfate, 3-10g/L of sodium citrate, 3-10g/L of dipotassium hydrogen phosphate, 3-10g/L of monopotassium hydrogen phosphate, 20g/L of agar, 10-30 mu g/ml of adenine and 10-30mmol/L of sucrose, wherein the pH value of the regenerated supplementary basic solid culture medium is 7.5; and/or the number of the groups of groups,

The protoplast stabilizing solution comprises the following components: sucrose 0.5-3.0mol/L, magnesium chloride 5-25mol/L and maleic acid 0.02-0.1mol/L, and the pH value of the protoplast stabilizing solution is 6.5.

Further, the strain sp1 is prepared according to the following steps:

separating the original 4-BA strain on a first plate screening culture medium after ultraviolet mutagenesis, performing expansion culture on a first seed culture medium, and finally fermenting through a first fermentation culture medium to select a strain sp1 capable of completely converting 4% of phytosterol.

Further, the method comprises the steps of,

the first seed medium comprises the following components (g/L): 5-20 parts of yeast powder, 10-20 parts of glucose, 3-10 parts of diammonium phosphate, 3-10 parts of ammonium citrate, 0.3-2.0 parts of magnesium sulfate heptahydrate, 0.3-2.0 parts of ferric ammonium citrate, 0.1-2.0 parts of calcium chloride and 0.2-2.0 parts of dichlord, wherein the pH value of the first seed culture medium is 7.5; and/or the number of the groups of groups,

the first plate screening medium comprises the following components (g/L): 10-20 parts of peptone, 10-20 parts of phytosterol, 3-10 parts of diammonium phosphate, 0.3-10 parts of magnesium sulfate heptahydrate, 0.3-2.0 parts of ferric ammonium citrate, 0.1-2.0 parts of calcium chloride and 20 parts of agar, wherein the pH value of the first plate screening culture medium is 7.5; and/or the number of the groups of groups,

the first fermentation medium comprises the following components (g/L): 20-50 parts of corn steep liquor, 5-20 parts of glycerol, 3-10 parts of diammonium hydrogen phosphate, 3-10 parts of sodium nitrate, 3-10 parts of ammonium citrate, 0.3-2.0 parts of magnesium sulfate heptahydrate, 0.3-2.0 parts of ferric ammonium citrate, 0.1-2.0 parts of calcium chloride, 80-160 parts of soybean oil, 10-30 parts of soybean lecithin, 5-20 parts of tween 80, 5-15 parts of bufomide and 40 parts of phytosterol, and the pH value of a first fermentation medium is 7.5.

Further, the strain sp2 is obtained by screening according to the following method:

separating the original 4-BA strain on a second plate screening culture medium after ultraviolet mutagenesis, performing expansion culture on a second seed culture medium, and finally performing experiments through a second fermentation culture medium to select a strain sp2 with the characteristic of high growth speed and seed culture time of 30-36 h.

Further, the method comprises the steps of,

the second seed medium comprises the following components (g/L): 15-25 parts of yeast powder, 15-30 parts of glucose, 3-10 parts of diammonium phosphate, 3-10 parts of ammonium citrate, 0.3-2.0 parts of magnesium sulfate heptahydrate, 0.3-2.0 parts of ferric ammonium citrate, 0.1-2.0 parts of calcium chloride and 0.2-2.0 parts of dichlord, and the pH value of the second seed culture medium is 7.5; and/or the number of the groups of groups,

the second plate screening medium comprises the following components (g/L): 10-25 parts of peptone, 10-20 parts of glucose, 3-10 parts of diammonium phosphate, 0.3-1.0 parts of magnesium sulfate heptahydrate, 0.3-1.0 parts of ferric ammonium citrate, 0.1-1.0 parts of calcium chloride and 20 parts of agar, wherein the pH value of the second plate screening culture medium is 7.5; and/or the number of the groups of groups,

the second fermentation medium comprises the following components (g/L): 30-50 parts of corn steep liquor, 5-20 parts of glycerol, 3-10 parts of diammonium hydrogen phosphate, 3-15 parts of sodium nitrate, 3-10 parts of ammonium citrate, 0.3-1.0 part of magnesium sulfate heptahydrate, 0.3-1.0 part of ferric ammonium citrate, 0.1-1.0 part of calcium chloride, 80-160 parts of soybean oil, 10-30 parts of soybean lecithin, 5-20 parts of tween 80, 5-15 parts of bufomide, 15 parts of phytosterol and 7.5 parts of a second fermentation medium.

The technical scheme of the invention has the following advantages:

1. the 4-BA paraincidentally Mycobacterium strain with the rapid growth and high transformation capacity provided by the invention has the high transformation capacity and the rapid growth capacity, is beneficial to the improvement of sterol yield, reduces the production cost and improves the production efficiency.

2. The method for producing the 4-BA paraincidentally mycobacterial strain with rapid growth and high transformation capacity provided by the invention improves the strain by utilizing a protoplast fusion technology, has no defect of distant hybridization unfused, and the screened strain has good stability, thus being a comparatively ideal directional microorganism strain breeding method. And genetic materials are fully recombined by utilizing a protoplast fusion technology, so that a fusion seed with strong transformation capacity and rapid growth is obtained, the strain growth speed can be effectively improved, the seed culture time and fermentation time are shortened, the power consumption is reduced, and the yield and production efficiency are improved.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the growth of a fungus body in the step of obtaining sp1 strain in example 1 of the present invention;

FIG. 2 is a graph showing the growth of a fungus body obtained by the step of obtaining an sp2 strain in example 1 of the present invention;

FIG. 3 is a graph showing the growth of a fungus body obtained in the step of obtaining sp1 strain in example 2 of the present invention;

FIG. 4 is a graph showing the growth of a fungus body obtained by the step of obtaining an sp2 strain in example 2 of the present invention;

FIG. 5 is a graph showing the growth of a fungus body obtained in the step of obtaining sp1 strain in example 3 of the present invention;

FIG. 6 is a graph showing the growth of a fungus body in the step of obtaining an sp2 strain in example 3 of the present invention.

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.

The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

The invention aims to overcome the defects of lower production efficiency and higher production cost caused by longer seed growth period of a 4-BA strain in the prior art, thereby providing a 4-BA paraincidentally mycobacterial strain with rapid growth and high transformation capacity and a preparation method thereof.

The invention provides a 4-BA pair accidental mycobacterium strain (Mycobacterium parafortuitum) with rapid growth and high transformation capacity, which is classified and named Mycobacterium parafortuitum BK047, and the preservation unit is China center for type culture collection, and the preservation date is as follows: year 2020, month 4, 23, deposit address: the preservation number of the Chinese university of Wuhan is CCTCC M2020076.

The preparation method of the 4-BA paraincidentally Mycobacterium strain with rapid growth and high transformation capacity provided by the invention comprises the following steps: carrying out protoplast fusion on a 4-BA strain sp1 with high transformation capacity and a 4-BA strain sp2 with rapid growth capacity in a polyethylene glycol system to obtain a fusion seed, and screening the fusion seed to obtain the 4-BA paraaccidental mycobacterium strain with rapid growth and high transformation capacity.

Wherein, 4-BA strain sp1 with high transformation capacity related by the invention is a paraincidentally Mycobacterium strain (Mycobacterium parafortuitum), the classification name is Mycobacterium parafortuitum BK045, the preservation unit is China center for type culture Collection, and the preservation date is: year 2020, month 4, 23, deposit address: the preservation number of the Chinese university of Wuhan is CCTCC M2020074.

4-BA strain sp2 paraincidentally Mycobacterium strain (Mycobacterium parafortuitum) having rapid growth ability, classified and named Mycobacterium parafortuitum BK046, deposit unit of China center for type culture collection, deposit date: year 2020, month 4, 23, deposit address: the preservation number of the Chinese university of Wuhan is CCTCC M2020075.

The polyethylene glycol is preferably PEG4000-6000 with a volume concentration of 40% -60%.

Specifically, the preparation method comprises the following steps:

1. acquisition of 4-BA Strain sp1 having high transformation Capacity

Separating and screening bacterial colonies with high growth speed from the original 4-BA strain on a first flat screening culture medium after ultraviolet mutagenesis, performing expansion culture on the bacterial colonies on a first seed culture medium, and finally fermenting the bacterial colonies by a first fermentation culture medium to select a bacterial strain sp1 capable of completely converting 4% of phytosterol.

The original 4-BA strain related by the invention is a paraincidentally mycobacterium strain (Mycobacterium parafortuitum), the classification name is Mycobacterium parafortuitum BK, the preservation unit is China center for type culture collection, and the preservation date is: address of deposit at 3/6/2017: the preservation number of the Chinese university of Wuhan is CCTCC M2017084.

Wherein the first seed medium used comprises the following components (g/L): 5-20 parts of yeast powder, 10-20 parts of glucose, 3-10 parts of diammonium phosphate, 3-10 parts of ammonium citrate, 0.3-2.0 parts of magnesium sulfate heptahydrate, 0.3-2.0 parts of ferric ammonium citrate, 0.1-2.0 parts of calcium chloride and 0.2-2.0 parts of dichlord, and the pH value is 7.5;

the first plate screening medium used included the following components (g/L): 10-20 parts of peptone, 10-20 parts of phytosterol, 3-10 parts of diammonium phosphate, 0.3-10 parts of magnesium sulfate heptahydrate, 0.3-2.0 parts of ferric ammonium citrate, 0.1-2.0 parts of calcium chloride, 20 parts of agar and pH value of 7.5; and/or the number of the groups of groups,

the first fermentation medium used was: 20-50 parts of corn steep liquor, 5-20 parts of glycerol, 3-10 parts of diammonium hydrogen phosphate, 3-10 parts of sodium nitrate, 3-10 parts of ammonium citrate, 0.3-2.0 parts of magnesium sulfate heptahydrate, 0.3-2.0 parts of ferric ammonium citrate, 0.1-2.0 parts of calcium chloride, 80-160 parts of soybean oil, 10-30 parts of soybean lecithin, 5-20 parts of tween, 5-15 parts of bufomide and 40 parts of phytosterol, wherein the pH value is 7.5.

The ultraviolet mutagenesis method comprises the following steps: the plate is placed under an ultraviolet lamp at a position of 10cm, the power of the ultraviolet lamp is 30w, and the irradiation time is 10min.

2. Acquisition of 4-BA Strain sp2 having Rapid growth Capacity

Screening and separating bacterial colonies with high growth speed from the original 4-BA strain on a second plate screening culture medium through ultraviolet mutagenesis, performing expansion culture on a second seed culture medium, and finally performing experiments through a second fermentation culture medium to select a strain sp2 with high growth speed and seed culture time of 30-36 h.

The second seed medium used included the following components (g/L): 15-25 parts of yeast powder, 15-30 parts of glucose, 3-10 parts of diammonium phosphate, 3-10 parts of ammonium citrate, 0.3-2.0 parts of magnesium sulfate heptahydrate, 0.3-2.0 parts of ferric ammonium citrate, 0.1-2.0 parts of calcium chloride and 0.2-2.0 parts of dichlord, and the pH value is 7.5; and/or the number of the groups of groups,

the second plate screening medium used included the following components (g/L): 10-25 parts of peptone, 10-20 parts of glucose, 3-10 parts of diammonium phosphate, 0.3-1.0 part of magnesium sulfate heptahydrate, 0.3-1.0 part of ferric ammonium citrate, 0.1-1.0 part of calcium chloride and 20 parts of agar, wherein the pH value is 7.5; and/or the number of the groups of groups,

the second fermentation medium used included the following components (g/L): 30-50 parts of corn steep liquor, 5-20 parts of glycerol, 3-10 parts of diammonium hydrogen phosphate, 3-15 parts of sodium nitrate, 3-10 parts of ammonium citrate, 0.3-1.0 part of magnesium sulfate heptahydrate, 0.3-1.0 part of ferric ammonium citrate, 0.1-1.0 part of calcium chloride, 80-160 parts of soybean oil, 10-30 parts of soybean lecithin, 5-20 parts of tween, 5-15 parts of bufomide and 15 parts of phytosterol, wherein the pH value is 7.5.

The ultraviolet mutagenesis method comprises the following steps: the plate is placed under an ultraviolet lamp at a position of 10cm, the power of the ultraviolet lamp is 30w, and the irradiation time is 10min.

3. Protoplast fusion of strains sp1 and sp2

(1) Preparation of protoplasts: culturing bacterial strain sp1 and bacterial strain sp2 on a liquid complete culture medium respectively, allowing cells to grow into a logarithmic growth phase, and removing walls to obtain protoplast suspensions of the bacterial strain sp1 and the bacterial strain sp2 respectively; wherein, the wall removing operation uses lysozyme for wall removing, the concentration of the used lysozyme solution is 60-100 mug/ml, and the wall removing operation comprises the step of enzymolysis for 35-50min at 36-40 ℃.

Preferably, the preparation of protoplasts specifically comprises the steps of:

(1) culturing the bacterial cells: the strain sp1 and sp2 monoclonal plates are respectively connected with a ring to a triangular flask filled with a liquid complete Culture Medium (CM), shaking culture is carried out for 14h at 36 ℃, 1ml of bacterial liquid is respectively connected with a 250ml conical flask filled with 20ml of liquid complete culture medium, shaking culture is carried out for 20h at 36 ℃, so that cells grow into logarithmic phase, 25 mug/ml kanamycin is respectively added, the final concentration is 0.5 mug/ml, and shaking culture is continued for 4h, thus obtaining bacterial liquid.

Wherein, the liquid complete culture medium comprises the following components: 10-15g/L of peptone, 10-15g/L of glucose, 5-10g/L of yeast powder, 5-10g/L of beef extract and 1-3g/L of sodium chloride; the pH of the liquid complete medium was 7.5.

(2) Collecting cells: 10ml of sp1 and sp2 bacteria solution was centrifuged at 4000r/min for 10min, and the supernatant was discarded, and the cells were suspended in phosphate buffer solution and centrifuged. The cells were then washed twice and suspended in 10ml of protoplastsIn volume stabilized liquid (SMM), about 10 per ml ⁸ -10 ⁹ Preferably living bacteria to obtain bacterial suspension.

Wherein the SMM solution comprises the following components: sucrose 0.5-3.0mol/L, magnesium chloride 5-25mol/L, maleic acid 0.02-0.1mol/L, and the pH value of SMM solution is 6.5.

(3) Total bacterial count determination: diluting 0.5ml of each bacterial liquid with physiological saline, and collecting 10 ^-5 、10 ^-6 、10 ^-7 1ml each (two plates per dilution), complete medium was poured and counted after incubation at 36℃for 24h, which is the total number of bacteria not treated with enzyme.

(4) Removing the wall: taking 5ml of bacterial suspension of each strain, adding 5ml of lysozyme solution, uniformly mixing, carrying out heat preservation treatment for 35min at 36 ℃ in a water bath, sampling at regular time, observing the formation of protoplasts by microscopic examination, centrifuging for 10min at 4000r/min when more than 95% of cells become spherical protoplasts, discarding supernatant, washing with a hypertonic buffer solution to remove enzymes, obtaining a protoplast suspension, suspending the protoplasts in 5ml of hypertonic buffer solution, and immediately measuring the residual bacterial count.

(5) Residual bacterial count measurement: taking 0.5ml of the protoplast suspension, diluting with sterile water to crack and kill protoplast, taking 10 ^-2 、10 ^-3 、10 ^-4 The dilutions were plated on complete medium plates at 36℃for 24-48h, and the colonies grown should be the remaining cells not lysed by the enzyme.

(6) Counting the number of cells remaining after the enzyme treatment: and the protoplast formation rate of the biparental strain was calculated as = (total number of cells not subjected to enzyme treatment—number of cells remaining after enzyme treatment)/total number of cells not subjected to enzyme treatment × 100%.

(2) Protoplast regeneration: carrying out regeneration culture on protoplast suspensions of the strain sp1 and the strain sp2 on a regeneration supplementing basic solid culture medium respectively to obtain regenerated protoplast suspensions of two parents;

the protoplast regeneration includes the following steps: the double-layer culture method comprises pouring regenerated supplemental basic solid culture medium (SMR) as bottom layer, collecting 0.5mL protoplast suspension, diluting with SMM solution10 ^-3 、10 ^-4 、10 ^-5 1ml of each diluent is added into the center of the bottom plate culture medium, then poured into the upper regenerated supplementary semi-solid culture medium, evenly mixed and cultured for 48 hours at 36 ℃ to obtain regenerated protoplast suspension.

Wherein the regeneration-supplemented minimal Solid Media (SMR) comprises the following components: 10-20g/L of glucose, 5-10g/L of ammonium sulfate, 3-10g/L of sodium citrate, 3-10g/L of dipotassium hydrogen phosphate, 3-10g/L of monopotassium hydrogen phosphate, 20g/L of agar, 10-30 mu g/ml of adenine and 10-30mmol/L of sucrose, wherein the pH value of the regenerated supplementary basic solid culture medium is 7.5.

Wherein the regeneration-supplemented semi-solid medium comprises the following components: 10-20g/L of glucose, 5-10g/L of ammonium sulfate, 3-10g/L of sodium citrate, 3-10g/L of dipotassium hydrogen phosphate, 3-10g/L of monopotassium hydrogen phosphate, 10g/L of agar, 10-30 mu g/ml of adenine and 10-30mmol/L of sucrose, wherein the pH value of the regenerated supplementary semisolid culture medium is 7.5.

(3) Protoplast fusion: respectively taking regenerated protoplast suspensions of the strain sp1 and the strain sp2, mixing, centrifuging, discarding supernatant, adding protoplast stabilizing solution and polyethylene glycol solution into the precipitate, and carrying out protoplast fusion;

protoplast fusion specifically includes the following steps: mixing 1mL of regenerated protoplast suspension of two parents, standing for 5min, centrifuging for 10min at 3500r/min, discarding supernatant, adding 0.2mL of SMM solution into the precipitate, mixing, adding 1.8mL of polyethylene glycol (PEG) solution, shaking gently, standing in a water bath at 36 ℃ for 2min, centrifuging for 10min at 3500r/min, collecting thallus, and suspending the precipitate in 2mL of SMM solution to obtain fusion solution.

(4) Detecting fusion seed:

taking 0.5mL of fusion solution, properly diluting with SMM solution, taking 0.1mL of bacterial solution, uniformly mixing with soft agar which is a regeneration supplementing basic culture medium and is sterilized and cooled to 50 ℃, rapidly pouring the mixture into a flat plate with a bottom layer which is the regeneration supplementing basic solid culture medium (SMR), culturing for 48 hours at 36 ℃, detecting the fusion seed, and transferring for passage.

(5) Screening of fusion:

selecting a fusion seed with stable genetic markers, which is a colony growing on a regenerated and supplemented minimal medium plate, preliminarily considering the colony as the fusion seed, accessing the fusion seed to a casein medium plate, and selecting the fusion seed with protease activity higher than that of a parent, wherein two conditions can occur after protoplast fusion: one is true fusion, i.e., the generation of heteronuclear diploids or haplotypes, and the other is the formation of heteronuclei with only mass coordination and no nuclear coordination. Both can form colonies on the regeneration minimal medium plates, but the former is stable, while the latter is unstable, so that the colonies will segregate into parent types in passage, so that several generations of segregation, purification and selection must be performed to obtain a true fusion, and finally strains with strong transformation capacity and high growth rate are obtained by screening.

(6) Fermentation tank test

The screened proper strain is subjected to a fermentation tank test, and the titer of the strain is determined.

Example 1

The example relates to a method for producing a 4-BA paraincidentally Mycobacterium strain with rapid growth and high transformation capacity, which comprises the following steps:

1. acquisition of 4-BA Strain sp1 having high transformation Capacity

(1) Separation

Taking an original 4-BA strain, carrying out gradient dilution on a bacterial suspension in a sterile super clean bench, coating the bacterial suspension on a first plate screening culture medium, placing the plate at a position of 10cm below an ultraviolet lamp, wherein the power of the ultraviolet lamp is 30w, the irradiation time is 10min, the culture is carried out at 31+/-1 ℃ for 7 days, observing once every 6 hours in the culture process, and marking a colony with a high growth speed.

The first plate screening medium included the following components (g/L): peptone 10, phytosterol 15, diammonium phosphate 6, magnesium sulfate heptahydrate 3.0, ferric ammonium citrate 0.8, calcium chloride 0.8, agar 20, and the pH of the first plate screening medium was 7.5.

(2) Preparation of monoclonal colonies

And selecting a single yellow colony on a first plate screening culture medium, marking the single yellow colony, and culturing for 7 days at 31+/-1 ℃ to obtain the single colony, wherein the diameter of the colony on the gradient dilution plate is larger, the growth speed is high, the morphology is better.

(3) Determination of cell growth curve

Inoculating mature monoclonal bacterial colony into a 100ml/500ml seed bottle, placing the seed bottle containing a first seed culture medium in a constant temperature shaking table with the rotation speed of 200r/min, shaking and culturing at 31+/-1 ℃, sampling every 6 hours, measuring absorbance at the wavelength of 600nm of a spectrophotometer, comparing with the first seed culture medium, plotting the absorbance with the bacterial growth time (h) as the abscissa and the absorbance as the ordinate, and drawing a bacterial growth curve, as shown in figure 1.

The first seed medium comprises the following components (g/L): 15 parts of yeast powder, 15 parts of glucose, 6 parts of diammonium phosphate, 6 parts of ammonium citrate, 1.0 part of magnesium sulfate heptahydrate, 0.8 part of ferric ammonium citrate, 0.8 part of calcium chloride, 1.0 part of dichlord and 7.5 parts of pH value.

(4) Fermentation shake flask investigation

Inoculating mature monoclonal colonies into a 30ml/250ml seed bottle, placing the seed bottle containing a first seed culture medium in a constant temperature shaking table with the rotating speed of 200r/min, shaking and culturing at 31+/-1 ℃ until the culture medium is in the mid-logarithmic phase, and then inoculating the seed bottle into a fermentation shaking bottle; the fermentation shake flask comprises a first fermentation culture medium, the concentration of the plant sterol in the fermentation shake flask is 4%, the temperature is 31+/-1 ℃, the plant sterol is cultured for 168 hours at 200r/min, sampling and detection are carried out, the strain with the optimal conversion is selected as a starting strain, sp1 is marked, the strain can completely convert 4% of plant sterol, and the generation amount of 4-BA is 2.25g.

The first fermentation medium included the following components (g/L): 30 parts of corn steep liquor, 10 parts of glycerol, 5 parts of diammonium phosphate, 5 parts of sodium nitrate, 6 parts of ammonium citrate, 1.0 part of magnesium sulfate heptahydrate, 1.0 part of ferric ammonium citrate, 0.8 part of calcium chloride, 120 parts of soybean oil, 20 parts of soybean phospholipids, 15 parts of tween 80, 40 parts of bufomide, 40 parts of phytosterol and 7.5 parts of pH value.

2. Acquisition of 4-BA Strain sp2 having Rapid growth Capacity

(1) Separation

Taking an original 4-BA strain, sucking 1ml of bacterial suspension from an glycerol pipe of the strain, carrying out gradient dilution in an aseptic super clean bench, coating the bacterial suspension on a second plate screening culture medium, placing the plate at a position of 10cm below an ultraviolet lamp, culturing for 4 days at 31+/-1 ℃ for 10min under the power of 30w, observing every 6h in the culturing process, and marking a colony with a high growth speed.

The second plate screening medium included the following components (g/L): peptone 15, glucose 15, diammonium phosphate 6, magnesium sulfate heptahydrate 0.6, ferric ammonium citrate 0.6, calcium chloride 0.5, agar 20 and pH 7.5.

(2) Preparation of monoclonal colonies

And selecting a single yellow colony on a second plate screening culture medium, marking the single yellow colony, and culturing for 4 days at 31+/-1 ℃ to obtain the single colony, wherein the diameter of the colony on the gradient dilution plate is larger, the growth speed is high, the morphology is better.

(3) Determination of cell growth curve

Inoculating mature monoclonal colony into a 100ml/500ml seed bottle, placing the seed bottle containing a second seed culture medium in a constant temperature shaking table with the rotation speed of 200r/min, shaking and culturing at 31+/-1 ℃, sampling every 6 hours, measuring absorbance at the wavelength of 600nm of a spectrophotometer, comparing with the second seed culture medium, plotting the absorbance with the bacterial growth time (h) as the abscissa and the absorbance as the ordinate, and drawing a bacterial growth curve, as shown in figure 2.

The second seed medium included the following components (g/L): 15 parts of yeast powder, 20 parts of glucose, 5 parts of diammonium phosphate, 6 parts of ammonium citrate, 0.6 part of magnesium sulfate heptahydrate, 0.6 part of ferric ammonium citrate, 0.3 part of calcium chloride, 0.8 part of dichlord and 7.5 parts of pH value.

(4) Fermentation shake flask investigation

Inoculating mature monoclonal bacterial colony into a seed bottle with the concentration of 30ml/250ml, placing the seed bottle containing a second seed culture medium in a constant-temperature shaking table with the rotation speed of 200r/min, shaking and culturing for 36h at 31+/-1 ℃, inoculating the seed bottle into a fermentation shaking bottle containing a second fermentation culture medium, culturing at 31+/-1 ℃ and 200r/min, selecting a bacterial strain with the concentration of the second seed liquid bacterial liquid being high and finally converting sterols completely as a starting bacterial strain at 36h, and marking the bacterial strain as sp2.

The second fermentation medium included the following components (g/L): corn steep liquor 40, glycerol 10, diammonium hydrogen phosphate 7, sodium nitrate 8, ammonium citrate 5, magnesium sulfate heptahydrate 0.5, ferric ammonium citrate 0.6, calcium chloride 0.1, soybean oil 120, soybean lecithin 20, tween 80 15, bufomide 10, phytosterol 15, pH 7.5

3. Protoplast fusion of strain sp1 with strain sp2

(1) Preparation of protoplasts:

culturing the bacterial cells: the strain sp1 and sp2 monoclonal plates are respectively connected with a ring to a triangular flask filled with a liquid complete Culture Medium (CM), shaking culture is carried out for 14h at 36 ℃, 1ml of bacterial liquid is respectively connected with a 250ml conical flask filled with 20ml of liquid complete culture medium, shaking culture is carried out for 20h at 36 ℃, so that cells grow into logarithmic growth phase, 25 mug/ml kanamycin is respectively added, the final concentration is 0.5 mug/ml, and shaking culture is continued for 4h.

Wherein, the liquid complete culture medium comprises the following components: 10g/L peptone, 15g/L glucose, 5g/L yeast powder, 5g/L beef extract and 1g/L sodium chloride; the pH of the liquid complete medium was 7.5.

Collecting cells: 10ml of each bacterial liquid was centrifuged at 4000r/min for 10min, the supernatant was discarded, and the bacterial cells were suspended in phosphate buffer and centrifuged. The cells were then washed twice and suspended in 10ml SMM, each ml containing about 10 ⁸ -10 ⁹ Live bacteria are preferable.

Total bacterial count determination: diluting 0.5ml of each bacterial liquid with physiological saline, and collecting 10 ^-5 、10 ^-6 、10 ^-7 1ml each (two plates per dilution), complete medium was poured and counted after incubation at 36℃for 24h, which is the total number of bacteria not treated with enzyme.

Removing the wall: taking 5ml of bacterial suspension of each strain, adding 5ml of lysozyme solution, uniformly mixing, carrying out heat preservation treatment for 35min at 36 ℃ in a water bath, sampling at regular time, observing the formation of protoplasts by microscopic examination, centrifuging for 10min at 4000r/min when more than 95% of cells become spherical protoplasts, discarding supernatant, washing with a hypertonic buffer solution to remove enzymes, obtaining a protoplast suspension, suspending the protoplasts in 5ml of hypertonic buffer solution, and immediately measuring the residual bacterial count.

Residual bacterial count measurement: taking 0.5ml of the protoplast suspension, diluting with sterile water to crack and kill protoplast, taking 10 ^-2 、10 ^-3 、10 ^-4 0.1mL of each dilution is coated on a complete culture medium plate, and the culture is carried out at 36 ℃ for 24-48 hours, so that the grown colonies are not grownRemaining cells of the enzymatic lysis.

Counting the number of cells remaining after the enzyme treatment: and the protoplast formation rate of the biparental strain was calculated as = (total number of cells not subjected to enzyme treatment—number of cells remaining after enzyme treatment)/total number of cells not subjected to enzyme treatment × 100%.

(2) Protoplast regeneration:

the double-layer culture method comprises pouring regenerated supplemental basic solid culture medium (SMR) as bottom layer, collecting 0.5mL protoplast suspension, diluting with SMM, collecting 10 ^-3 、10 ^-4 、10 ^-5 1ml of each diluent is added into the center of the bottom plate culture medium, then poured into the upper regenerated supplementary semi-solid culture medium, evenly mixed and cultured for 48 hours at 36 ℃ to obtain regenerated protoplast suspension. The protoplast regeneration rates of the amphipathic strains were calculated separately and the average thereof was calculated.

Wherein the regeneration supplementing basic solid culture medium comprises the following components: glucose 20g/L, ammonium sulfate 5g/L, sodium citrate 3g/L, dipotassium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 3g/L, agar 20g/L, adenine 20 mu g/ml and sucrose 10mmol/L, wherein the pH value of the regenerated supplemental basic solid culture medium is 7.5.

The SMM solution comprises the following components: sucrose 2.0mol/L, magnesium chloride 20mol/L, maleic acid 0.06mol/L, and the pH value of the SMM solution is 6.5.

The formation rate of the two protoplasts was about 85% and the regeneration rate was about 58%.

(3) Protoplast fusion:

mixing 1ml of regenerated protoplast suspension of two parents, standing for 5min, centrifuging for 10min at 3500r/min, discarding supernatant, adding 0.2ml of SMM solution into the precipitate, mixing, adding 1.8ml of PEG solution, shaking gently, standing in a water bath at 36 ℃ for 2min, centrifuging for 10min at 3500r/min, collecting thallus, suspending the precipitate in 2ml of SMM solution, and fusing protoplast to obtain fusion solution.

(4) Detecting fusion seed:

taking 0.5mL of fusion solution, properly diluting with SMM solution, taking 0.1mL of bacterial solution, uniformly mixing with soft agar of a regeneration supplementing basic culture medium which is sterilized and cooled to 50 ℃, rapidly pouring the mixture into a flat plate with the bottom layer being the regeneration supplementing basic culture medium, culturing for 48 hours at 36 ℃, detecting the fusion seed, and transferring for passage.

(5) Screening of fusion:

selecting a fusion seed with stable genetic markers, which is a colony growing on a regenerated and supplemented minimal medium plate, preliminarily considering the colony as the fusion seed, accessing the fusion seed to a casein medium plate, and selecting the fusion seed with protease activity higher than that of a parent, wherein two conditions can occur after protoplast fusion: one is true fusion, i.e., the generation of heteronuclear diploids or haplotypes, the other occurs only with mass coordination, and no nuclear coordination forms heteronuclear. Both can form colonies on the regeneration minimal medium plates, but the former is stable, while the latter is unstable, so that the colonies will segregate into parent types in passage, so that several generations of segregation, purification and selection must be performed to obtain a true fusion, and finally strains with strong transformation capacity and high growth rate are obtained by screening.

Randomly selecting a plurality of strains which grow after fusion, preparing a mother inclined plane, inoculating a shake flask for seed culture after culture maturation, firstly selecting the strain with the largest bacterial body quantity at 36 hours, and totally screening 15 strains (RJC 1-RJC 15); the 15 strains were subjected to fermentation transformation test screening, and after 7 days of culture, the transformation titers were determined, and the results of the examination of the fusion transformation titers are shown in Table 1.

Example 2

1. Acquisition of 4-BA Strain sp1 having high transformation Capacity

(1) Separation

Taking an original 4-BA strain, carrying out gradient dilution on a bacterial suspension in a sterile super clean bench, coating the bacterial suspension on a first plate screening culture medium, placing the plate at a position of 10cm below an ultraviolet lamp, wherein the power of the ultraviolet lamp is 30w, the irradiation time is 10min, the culture is carried out at 31+/-1 ℃ for 7 days, observing once every 6 hours in the culture process, and marking a colony with a high growth speed.

The first plate screening medium included the following components (g/L): peptone 5, phytosterol 10, diammonium phosphate 3, magnesium sulfate heptahydrate 0.3, ferric ammonium citrate 0.3, calcium chloride 0.1, agar 20, and the pH of the first plate screening medium was 7.5.

(2) Preparation of monoclonal colonies

And selecting a single yellow colony on a first plate screening culture medium, marking the single yellow colony, and culturing for 7 days at 31+/-1 ℃ to obtain the single colony, wherein the diameter of the colony on the gradient dilution plate is larger, the growth speed is high, the morphology is better.

(3) Determination of cell growth curve

Inoculating mature monoclonal colony into a 100ml/500ml seed bottle, placing the seed bottle containing a first seed culture medium in a constant temperature shaking table with the rotation speed of 200r/min, shaking and culturing at 31+/-1 ℃, sampling every 6 hours, measuring absorbance at the wavelength of 600nm of a spectrophotometer, comparing with the first seed culture medium, plotting the absorbance with the bacterial growth time (h) as the abscissa and the absorbance as the ordinate, and drawing a bacterial growth curve, as shown in figure 3.

The first seed medium comprises the following components (g/L): yeast powder 5, glucose 10, diammonium phosphate 3, ammonium citrate 3, magnesium sulfate heptahydrate 0.3, ferric ammonium citrate 0.3, calcium chloride 0.1, dichlord 0.2 and pH 7.5.

(4) Fermentation shake flask investigation

Inoculating mature monoclonal colonies into a 30ml/250ml seed bottle, placing the seed bottle containing a first seed culture medium in a constant temperature shaking table with the rotating speed of 200r/min, shaking and culturing at 31+/-1 ℃ until the culture medium is in the mid-logarithmic phase, and then inoculating the seed bottle into a fermentation shaking bottle; the fermentation shake flask comprises a first fermentation culture medium, the concentration of the plant sterol in the fermentation shake flask is 4%, the temperature is 31+/-1 ℃, the plant sterol is cultured for 168 hours at 200r/min, sampling and detection are carried out, the strain with the optimal conversion is selected as a starting strain, sp1 is marked, the strain can completely convert 4% of plant sterol, and the generation amount of 4-BA is 2.15g.

The first fermentation medium included the following components (g/L): corn steep liquor 20, glycerol 5, diammonium phosphate 3, sodium nitrate 3, ammonium citrate 3, magnesium sulfate heptahydrate 0.3, ferric ammonium citrate 0.3, calcium chloride 0.1, soybean oil 80, soybean lecithin 10, tween 80 5, bufomide 5, phytosterol 40 and pH 7.5.

2. Acquisition of 4-BA Strain sp2 having Rapid growth Capacity

(1) Separation

Taking an original 4-BA strain, sucking 1ml of bacterial suspension from an glycerol pipe of the strain, carrying out gradient dilution in an aseptic super clean bench, coating the bacterial suspension on a second plate screening culture medium, placing the plate at a position of 10cm below an ultraviolet lamp, culturing for 4 days at 31+/-1 ℃ for 10min under the power of 30w, observing every 6h in the culturing process, and marking a colony with a high growth speed.

The second plate screening medium included the following components (g/L): peptone 10, glucose 1,0, diammonium phosphate 3, magnesium sulfate heptahydrate 0.3, ferric ammonium citrate 0.3, calcium chloride 0.1, agar 20, and pH 7.5.

(2) Preparation of monoclonal colonies

And selecting a single yellow colony on a second plate screening culture medium, marking the single yellow colony, and culturing for 4 days at 31+/-1 ℃ to obtain the single colony, wherein the diameter of the colony on the gradient dilution plate is larger, the growth speed is high, the morphology is better.

(3) Determination of cell growth curve

Inoculating mature monoclonal colonies into a 100ml/500ml seed bottle, placing the seed bottle containing a second seed culture medium in a constant temperature shaking table with the rotation speed of 200r/min, shaking and culturing at 31+/-1 ℃, sampling every 6 hours, measuring absorbance at the wavelength of 600nm of a spectrophotometer, comparing with the second seed culture medium, plotting the absorbance with the bacterial growth time (h) as an abscissa and the absorbance as an ordinate, and drawing a bacterial growth curve, as shown in figure 4.

The second seed medium included the following components (g/L): yeast powder 15, glucose 15, diammonium phosphate 3, ammonium citrate 3, magnesium sulfate heptahydrate 0.3, ferric ammonium citrate 0.3, calcium chloride 0.1, dichlord 0.2 and pH 7.5.

(4) Fermentation shake flask investigation

Inoculating mature monoclonal bacterial colony into a seed bottle with the concentration of 30ml/250ml, placing the seed bottle containing a second seed culture medium in a constant-temperature shaking table with the rotation speed of 200r/min, shaking and culturing for 36h at 31+/-1 ℃, inoculating the seed bottle into a fermentation shaking bottle containing a second fermentation culture medium, culturing at 31+/-1 ℃ and 200r/min, selecting a bacterial strain with the concentration of the second seed liquid bacterial liquid being high and finally converting sterols completely as a starting bacterial strain at 36h, and marking the bacterial strain as sp2.

The second fermentation medium included the following components (g/L): corn steep liquor 30, glycerol 5, diammonium phosphate 3, sodium nitrate 3, ammonium citrate 3, magnesium sulfate heptahydrate 0.3, ferric ammonium citrate 0.3, calcium chloride 0.1, soybean oil 80, soybean lecithin 10, tween 80 5, bufomide 5, phytosterol 15, pH 7.5

3. Protoplast fusion of strain sp1 with strain sp2

(1) Preparation of protoplasts:

culturing the bacterial cells: the strain sp1 and sp2 monoclonal plates are respectively connected with a ring to a triangular flask filled with a liquid complete Culture Medium (CM), shaking culture is carried out for 14h at 36 ℃, 1ml of bacterial liquid is respectively connected with a 250ml conical flask filled with 20ml of liquid complete culture medium, shaking culture is carried out for 20h at 36 ℃, so that cells grow into logarithmic growth phase, 25 mug/ml kanamycin is respectively added, the final concentration is 0.5 mug/ml, and shaking culture is continued for 4h.

Wherein, the liquid complete culture medium comprises the following components: 12g/L peptone, 15g/L glucose, 7g/L yeast powder, 7g/L beef extract and 1g/L sodium chloride; the pH of the liquid complete medium was 7.5.

Collecting cells: 10ml of each bacterial liquid was centrifuged at 4000r/min for 10min, the supernatant was discarded, and the bacterial cells were suspended in phosphate buffer and centrifuged. The cells were then washed twice and suspended in 10ml SMM, each ml containing about 10 ⁸ -10 ⁹ Live bacteria are preferable.

Total bacterial count determination: diluting 0.5ml of each bacterial liquid with physiological saline, and collecting 10 ^-5 、10 ^-6 、10 ^-7 1ml each (two plates per dilution), complete medium was poured and counted after incubation at 36℃for 24h, which is the total number of bacteria not treated with enzyme.

Removing the wall: taking 5ml of bacterial suspension of each strain, adding 5ml of lysozyme solution, uniformly mixing, carrying out heat preservation treatment for 35min at 36 ℃ in a water bath, sampling at regular time, observing the formation of protoplasts by microscopic examination, centrifuging for 10min at 4000r/min when more than 95% of cells become spherical protoplasts, discarding supernatant, washing with hypertonic buffer solution to remove enzyme, suspending the protoplasts in 5ml of hypertonic buffer solution to obtain protoplast suspension, and immediately measuring the residual bacterial count.

Residual bacterial count measurement: taking 0.5ml of the protoplast suspension, diluting with sterile water to crack and kill protoplast, taking 10 ^-2 、10 ^-3 、10 ^-4 The dilutions were plated on complete medium plates at 36℃for 24-48h, and the colonies grown should be the remaining cells not lysed by the enzyme.

Counting the number of cells remaining after the enzyme treatment: and the protoplast formation rate of the biparental strain was calculated as = (total number of cells not subjected to enzyme treatment—number of cells remaining after enzyme treatment)/total number of cells not subjected to enzyme treatment × 100%.

(2) Protoplast regeneration:

the double-layer culture method comprises pouring regenerated supplemental basic solid culture medium (SMR) as bottom layer, collecting 0.5mL protoplast suspension, diluting with SMM, collecting 10 ^-3 、10 ^-4 、10 ^-5 1ml of each diluent is added into the center of the bottom plate culture medium, then poured into the upper regenerated supplementary semi-solid culture medium, evenly mixed and cultured for 48 hours at 36 ℃ to obtain regenerated protoplast suspension. The protoplast regeneration rates of the amphipathic strains were calculated separately and the average thereof was calculated.

Wherein the regeneration supplementing basic solid culture medium comprises the following components: glucose 20g/L, ammonium sulfate 5g/L, sodium citrate 3g/L, dipotassium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 3g/L, agar 20g/L, adenine 20 mu g/ml and sucrose 10mmol/L, wherein the pH value of the regenerated supplemental basic solid culture medium is 7.5.

The SMM solution comprises the following components: sucrose 0.8mol/L, magnesium chloride 5mol/L, maleic acid 0.02mol/L, and the pH of the SMM solution was 6.5.

The formation rate of the two protoplasts was about 78%, and the regeneration rate was about 56%.

(3) Protoplast fusion:

mixing 1ml of regenerated protoplast suspension of two parents, standing for 5min, centrifuging for 10min at 3500r/min, discarding supernatant, adding 0.2ml of SMM solution into the precipitate, mixing, adding 1.8ml of PEG solution, shaking gently, standing in a water bath at 36 ℃ for 2min, centrifuging for 10min at 3500r/min, collecting thallus, suspending the precipitate in 2ml of SMM solution, and fusing protoplast to obtain fusion solution.

(4) Detecting fusion seed:

taking 0.5mL of fusion solution, properly diluting with SMM solution, taking 0.1mL of bacterial solution, uniformly mixing with soft agar of a regeneration supplementing basic culture medium which is sterilized and cooled to 50 ℃, rapidly pouring the mixture into a flat plate with the bottom layer being the regeneration supplementing basic culture medium, culturing for 48 hours at 36 ℃, detecting the fusion seed, and transferring for passage.

(5) Screening of fusion:

selecting a fusion seed with stable genetic markers, which is a colony growing on a regenerated and supplemented minimal medium plate, preliminarily considering the colony as the fusion seed, accessing the fusion seed to a casein medium plate, and selecting the fusion seed with protease activity higher than that of a parent, wherein two conditions can occur after protoplast fusion: one is true fusion, i.e., the generation of heteronuclear diploids or haplotypes, the other occurs only with mass coordination, and no nuclear coordination forms heteronuclear. Both can form colonies on the regeneration minimal medium plates, but the former is stable, while the latter is unstable, so that the colonies will segregate into parent types in passage, so that several generations of segregation, purification and selection must be performed to obtain a true fusion, and finally strains with strong transformation capacity and high growth rate are obtained by screening.

Randomly selecting a plurality of strains which grow after fusion, preparing a mother inclined plane, inoculating a shake flask for seed culture after culture maturation, firstly selecting the strain with the largest bacterial body quantity at 36 hours, and totally screening 15 strains (RJC 16-RJC 30); the 15 strains were subjected to fermentation transformation test screening, and after 7 days of culture, the transformation titers were determined, and the results of the examination of the fusion transformation titers are shown in Table 1.

Example 3

1. Acquisition of 4-BA Strain sp1 having high transformation Capacity

(1) Separation

Taking an original 4-BA strain, carrying out gradient dilution on a bacterial suspension in a sterile super clean bench, coating the bacterial suspension on a first plate screening culture medium, placing the plate at a position of 10cm below an ultraviolet lamp, wherein the power of the ultraviolet lamp is 30w, the irradiation time is 10min, the culture is carried out at 31+/-1 ℃ for 7 days, observing once every 6 hours in the culture process, and marking a colony with a high growth speed.

The first plate screening medium included the following components (g/L): peptone 20, phytosterol 20, diammonium phosphate 10, magnesium sulfate heptahydrate 9, ferric ammonium citrate 2.0, calcium chloride 2.0, agar 20, and the pH of the first plate screening medium was 7.5.

(2) Preparation of monoclonal colonies

And selecting a single yellow colony on a first plate screening culture medium, marking the single yellow colony, and culturing for 7 days at 31+/-1 ℃ to obtain the single colony, wherein the diameter of the colony on the gradient dilution plate is larger, the growth speed is high, the morphology is better.

(3) Determination of cell growth curve

Inoculating mature monoclonal colony into a 100ml/500ml seed bottle, placing the seed bottle containing a first seed culture medium in a constant temperature shaking table with the rotation speed of 200r/min, shaking and culturing at 31+/-1 ℃, sampling every 6 hours, measuring absorbance at the wavelength of 600nm of a spectrophotometer, comparing with the first seed culture medium, plotting the absorbance with the bacterial growth time (h) as the abscissa and the absorbance as the ordinate, and drawing a bacterial growth curve, as shown in figure 5.

The first seed medium comprises the following components (g/L): yeast powder 20, glucose 20, diammonium phosphate 10, ammonium citrate 10, magnesium sulfate heptahydrate 2.0, ferric ammonium citrate 2.0, calcium chloride 2.0, dichlord 0.2 and pH 7.5.

(4) Fermentation shake flask investigation

Inoculating mature monoclonal colonies into a 30ml/250ml seed bottle, placing the seed bottle containing a first seed culture medium in a constant temperature shaking table with the rotating speed of 200r/min, shaking and culturing at 31+/-1 ℃ until the culture medium is in the mid-logarithmic phase, and then inoculating the seed bottle into a fermentation shaking bottle; the fermentation shake flask comprises a first fermentation culture medium, the concentration of the plant sterol in the fermentation shake flask is 4%, the temperature is 31+/-1 ℃, the plant sterol is cultured for 168 hours at 200r/min, sampling and detection are carried out, the strain with the optimal conversion is selected as a starting strain, sp1 is marked, the strain can completely convert 4% of plant sterol, and the generation amount of 4-BA is 2.32g.

The first fermentation medium included the following components (g/L): corn steep liquor 50, glycerin 20, diammonium phosphate 10, sodium nitrate 10, ammonium citrate 10, magnesium sulfate heptahydrate 2.0, ferric ammonium citrate 2.0, calcium chloride 2.0, soybean oil 160, soybean lecithin 30, tween 80 20, dichlord 15, phytosterol 40 and pH 7.5.

2. Acquisition of 4-BA Strain sp2 having Rapid growth Capacity

(1) Separation

Taking an original 4-BA strain, sucking 1ml of bacterial suspension from an glycerol pipe of the strain, carrying out gradient dilution in an aseptic super clean bench, coating the bacterial suspension on a second plate screening culture medium, placing the plate at a position of 10cm below an ultraviolet lamp, culturing for 4 days at 31+/-1 ℃ for 10min under the power of 30w, observing every 6h in the culturing process, and marking a colony with a high growth speed.

The second plate screening medium included the following components (g/L): 25 parts of peptone, 20 parts of glucose, 10 parts of diammonium phosphate, 1.0 part of magnesium sulfate heptahydrate, 1.0 part of ferric ammonium citrate, 1.0 part of calcium chloride, 20 parts of agar and 7.5 parts of pH value.

(2) Preparation of monoclonal colonies

And selecting a single yellow colony on a second plate screening culture medium, marking the single yellow colony, and culturing for 4 days at 31+/-1 ℃ to obtain the single colony, wherein the diameter of the colony on the gradient dilution plate is larger, the growth speed is high, the morphology is better.

(3) Determination of cell growth curve

Inoculating mature monoclonal colony into a 100ml/500ml seed bottle, placing the seed bottle containing a second seed culture medium in a constant temperature shaking table with the rotation speed of 200r/min, shaking and culturing at 31+/-1 ℃, sampling every 6 hours, measuring absorbance at the wavelength of 600nm of a spectrophotometer, comparing with the second seed culture medium, plotting the absorbance with the bacterial growth time (h) as the abscissa and the absorbance as the ordinate, and drawing a bacterial growth curve, as shown in figure 6.

The second seed medium included the following components (g/L): 25 parts of yeast powder, 30 parts of glucose, 10 parts of diammonium phosphate, 10 parts of ammonium citrate, 2.0 parts of magnesium sulfate heptahydrate, 2.0 parts of ferric ammonium citrate, 2.0 parts of calcium chloride, 0.2 parts of dichlord and 7.5 parts of pH value.

(4) Fermentation shake flask investigation

Inoculating mature monoclonal bacterial colony into a seed bottle with the concentration of 30ml/250ml, placing the seed bottle containing a second seed culture medium in a constant-temperature shaking table with the rotation speed of 200r/min, shaking and culturing for 36h at 31+/-1 ℃, inoculating the seed bottle into a fermentation shaking bottle containing a second fermentation culture medium, culturing at 31+/-1 ℃ and 200r/min, selecting a bacterial strain with the concentration of the second seed liquid bacterial liquid being high and finally converting sterols completely as a starting bacterial strain at 36h, and marking the bacterial strain as sp2.

The second fermentation medium included the following components (g/L): corn steep liquor 50, glycerin 20, diammonium phosphate 10, sodium nitrate 15, ammonium citrate 3, magnesium sulfate heptahydrate 1.0, ferric ammonium citrate 1.0, calcium chloride 1.0, soybean oil 160, soybean lecithin 30, tween 80 20, bufomide 15, phytosterol 15, pH 7.5

3. Protoplast fusion of strain sp1 with strain sp2

(1) Preparation of protoplasts:

culturing the bacterial cells: the strain sp1 and sp2 monoclonal plates are respectively connected with a ring to a triangular flask filled with a liquid complete Culture Medium (CM), shaking culture is carried out for 14h at 36 ℃, 1ml of bacterial liquid is respectively connected with a 250ml conical flask filled with 20ml of liquid complete culture medium, shaking culture is carried out for 20h at 36 ℃, so that cells grow into logarithmic growth phase, 25 mug/ml kanamycin is respectively added, the final concentration is 0.5 mug/ml, and shaking culture is continued for 4h.

Wherein, the liquid complete culture medium comprises the following components: 12g/L peptone, 15g/L glucose, 7g/L yeast powder, 7g/L beef extract and 1g/L sodium chloride; the pH of the liquid complete medium was 7.5.

Collecting cells: 10ml of each bacterial liquid was centrifuged at 4000r/min for 10min, the supernatant was discarded, and the bacterial cells were suspended in phosphate buffer and centrifuged. The cells were then washed twice and suspended in 10ml SMM, each ml containing about 10 ⁸ -10 ⁹ Live bacteria are preferable.

Total average measurement: diluting 0.5ml of each bacterial liquid with physiological saline, and collecting 10 ^-5 、10 ^-6 、10 ^-7 1ml each (two plates per dilution), complete medium was poured and counted after incubation at 36℃for 24h, which is the total number of bacteria not treated with enzyme.

Removing the wall: taking 5ml of bacterial suspension of each strain, adding 5ml of lysozyme solution, uniformly mixing, carrying out heat preservation treatment for 35min at 36 ℃ in a water bath, sampling at regular time, observing the formation of protoplasts by microscopic examination, centrifuging for 10min at 4000r/min when more than 95% of cells become spherical protoplasts, discarding supernatant, washing with a hypertonic buffer solution to remove enzymes, suspending the protoplasts in 5ml of the hypertonic buffer solution to obtain protoplast suspension, and immediately measuring the residual bacterial count.

Residual bacterial count measurement: taking 0.5ml of the protoplast suspension, diluting with sterile water to crack and kill protoplast, taking 10 ^-2 、10 ^-3 、10 ^-4 The dilutions were plated on complete medium plates at 36℃for 24-48h, and the colonies grown should be the remaining cells not lysed by the enzyme.

Counting the number of cells remaining after the enzyme treatment: and the protoplast formation rate of the biparental strain was calculated as = (total number of cells not subjected to enzyme treatment—number of cells remaining after enzyme treatment)/total number of cells not subjected to enzyme treatment × 100%.

(2) Protoplast regeneration:

the double-layer culture method comprises pouring regenerated supplemental basic solid culture medium (SMR) as bottom layer, collecting 0.5mL protoplast suspension, diluting with SMM, collecting 10 ^-3 、10 ^-4 、10 ^-5 1ml of each diluent is added into the center of the bottom plate culture medium, then poured into the upper regenerated supplementary semi-solid culture medium, evenly mixed and cultured for 48 hours at 36 ℃ to obtain regenerated protoplast suspension. The protoplast regeneration rates of the amphipathic strains were calculated separately and the average thereof was calculated.

Wherein the regeneration supplementing basic solid culture medium comprises the following components: glucose 20g/L, ammonium sulfate 5g/L, sodium citrate 3g/L, dipotassium hydrogen phosphate 3g/L, potassium dihydrogen phosphate 3g/L, agar 20g/L, adenine 20 mu g/ml and sucrose 10mmol/L, wherein the pH value of the regenerated supplemental basic solid culture medium is 7.5.

The SMM solution comprises the following components: sucrose 3.0mol/L, magnesium chloride 25mol/L, maleic acid 0.1mol/L, and the pH of the SMM solution was 6.5.

The formation rate of the two protoplasts was 89% or so, and the regeneration rate was 61% or so.

(3) Protoplast fusion:

mixing 1ml of regenerated protoplast suspension of two parents, standing for 5min, centrifuging for 10min at 3500r/min, discarding supernatant, adding 0.2ml of SMM solution into the precipitate, mixing, adding 1.8ml of PEG solution, shaking gently, standing in a water bath at 36 ℃ for 2min, centrifuging for 10min at 3500r/min, collecting thallus, suspending the precipitate in 2ml of SMM solution, and fusing protoplast to obtain fusion solution.

(4) Detecting fusion seed:

taking 0.5mL of fusion solution, properly diluting with SMM solution, taking 0.1mL of bacterial solution, uniformly mixing with soft agar of a regeneration supplementing basic culture medium which is sterilized and cooled to 50 ℃, rapidly pouring the mixture into a flat plate with the bottom layer being the regeneration supplementing basic culture medium, culturing for 48 hours at 36 ℃, detecting the fusion seed, and transferring for passage.

(5) Screening of fusion:

selecting a fusion seed with stable genetic markers, which is a colony growing on a regenerated and supplemented minimal medium plate, preliminarily considering the colony as the fusion seed, accessing the fusion seed to a casein medium plate, and selecting the fusion seed with protease activity higher than that of a parent, wherein two conditions can occur after protoplast fusion: one is true fusion, i.e., the generation of heteronuclear diploids or haplotypes, the other occurs only with mass coordination, and no nuclear coordination forms heteronuclear. Both can form colonies on the regeneration minimal medium plates, but the former is stable, while the latter is unstable, so that the colonies will segregate into parent types in passage, so that several generations of segregation, purification and selection must be performed to obtain a true fusion, and finally strains with strong transformation capacity and high growth rate are obtained by screening.

Randomly selecting a plurality of strains which grow after fusion, preparing a mother inclined plane, inoculating a shake flask for seed culture after culture maturation, firstly selecting the strain with the largest bacterial body quantity at 36 hours, and totally screening 15 strains (RJC 31-RJC 45); the 15 strains were subjected to fermentation transformation test screening, and after 7 days of culture, the transformation titers were determined, and the results of the examination of the fusion transformation titers are shown in Table 1.

Fusions potency investigation

1. Fermentation shake flask transformation verification test:

the conversion titers of strains RJC1-RJC45 are shown in Table 1.

Wherein, the seed culture medium for screening out the RJC1-RJC45 strain comprises the following components (g/L): yeast powder 10, glucose 12, diammonium phosphate 3, ammonium citrate 3, magnesium sulfate heptahydrate 0.7, ferric ammonium citrate 0.7, calcium chloride 0.1 and dichlord 0.4, and the pH value is 7.5.

The transformation medium used in the fermentation transformation test comprises the following components (g/L): corn steep liquor 40, glycerin 10, diammonium hydrogen phosphate 5, sodium nitrate 5, ammonium citrate 6, magnesium sulfate heptahydrate 0.5, ferric ammonium citrate 1.0, calcium chloride 0.1, soybean oil 160, soybean lecithin 15, tween 80 8, phytosterol 40, bufomide 12 and pH 7.5.

The fermentation conversion is tested by using a shaking flask, the rotation speed of a shaking table is 200rmp, the culture temperature is 32 ℃, and the inoculation amount is 12%.

TABLE 1 results of the fusion sub-investigation

Among them, the fusion RJC40 was most preferred, 4% of plant sterols could be completely converted, and the amount of 4-BA produced was 2.67g.

2. Genetic stability study test:

to determine the genetic stability of the strain RJC40, the strain was subjected to serial passage on a seed culture medium for 10 times, each strain was transferred to a shake flask containing a transformation medium for fermentation culture, the seed growth period was observed, the 4-BA production amount was measured after 7d fermentation culture, and the results of the genetic stability investigation are shown in Table 2.

Wherein, the genetic stability investigation test seed culture medium comprises the following components (g/L): yeast powder 10, glucose 12, diammonium phosphate 3, ammonium citrate 3, magnesium sulfate heptahydrate 0.7, ferric ammonium citrate 0.7, calcium chloride 0.1 and dichlord 0.4, and the pH value is 7.5.

Genetic stability investigation test transformation medium comprises the following components (g/L): corn steep liquor 40, glycerin 10, diammonium hydrogen phosphate 5, sodium nitrate 5, ammonium citrate 6, magnesium sulfate heptahydrate 0.5, ferric ammonium citrate 1.0, calcium chloride 0.1, soybean oil 160, soybean lecithin 15, tween 80 8, phytosterol 40, bufomide 12 and pH 7.5.

Genetic stability investigation test is carried out by using a shaking flask, the rotation speed of a shaking table is 200rmp, the culture temperature is 32 ℃, and the inoculation amount is 12%.

TABLE 2 results of fusion RJC40 serial passage experiments

Number of passages	4-BA production amount/g	Number of passages	4-BA production amount/g
				W1	2.59	W6	2.71
W2	2.62	W7	2.64
				W3	2.71	W8	2.69
W4	2.62	W9	2.68
				W5	2.63	W10	2.57

RJC40 has stable continuous passage character, consistent seed growth period and fermentation titer, and can be determined to be a new strain with strong transformation capability and high growth speed and genetic stability.

3. Fermentation tank conversion test:

fermentation tank transformation test strain RJC40 is tested by using NBS BioFlo 5000 and NBS BioFlo 110 fermentation tanks, the primary seed inoculation amount is 1%, and the strain RJC40 is cultured for 48 hours and then is transferred into a secondary seed tank; the inoculation amount of the secondary seeds is 10 percent, and the growth period of the secondary seed tank is 36 hours.

Wherein, the primary seed culture medium and the secondary seed culture medium comprise the following components (g/L): yeast powder 10, glucose 12, diammonium phosphate 3, ammonium citrate 3, magnesium sulfate heptahydrate 0.7, ferric ammonium citrate 0.7, calcium chloride 0.1 and dichlord 0.4, and the pH value is 7.5.

The primary seeds are cultivated by shaking bottles, the rotation speed of a shaking table is 200rmp, and the cultivation temperature is 32 ℃; the secondary seeds were cultured in NBS BioFlo 110 10L fermentor at a stirring speed of 200rmp at 32℃and aeration rate of 0.2vvm.

The fermenter test medium comprises the following components (g/L): corn steep liquor 40, glycerin 10, diammonium hydrogen phosphate 5, sodium nitrate 5, ammonium citrate 6, magnesium sulfate heptahydrate 0.5, ferric ammonium citrate 1.0, calcium chloride 0.1, soybean oil 160, soybean lecithin 15, tween 80 8, phytosterol 40, bufomide 12 and pH 7.5.

The fermenter test was performed using an NBS BioFlo 5000 80L fermenter with a stirring speed of 500rmp, a cultivation temperature of 32℃and an aeration rate of 0.5vvm.

The seed of the first-stage seed tank is shortened by 24 hours compared with the original strain sp1, the seed of the second-stage seed tank is shortened by 36 hours compared with the original strain sp1, the yield of 4-BA is measured by tank discharge, the substrate concentration is 4% of phytosterol, the yield of 4-BA is 2.86g, the conversion time is 124 hours, the two data of the original strain are 2.28g and 208 hours, the advantages of the new strain are obvious, and the successful screening of the strain RJC40 can be determined.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims (1)

1. A strain (Mycobacterium parafortuitum) of Mycobacterium paraincidentally with rapid growth and high transformation capability is classified and named Mycobacterium parafortuitum BK047, the preservation unit is China center for type culture collection, and the preservation number is CCTCC M2020076.

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