TWI472622B - Method for producing brazzein by using controllably acid-inducible system - Google Patents

Description

Method for producing sweet protein using a controllable acid induction system

This invention relates to a method of making a sweet protein, and more particularly to a method of making a sweet protein using a controlled acid induction system.

Lactic acid bacteria are a group of fairly complex bacteria that are widely distributed in nature and can be fermented with carbohydrates to produce lactic acid. It has been widely used in dairy products, food processing or preservation since ancient times. It is also regarded as a food grade strain, and it is one of the most widely used and economically effective industrial bacteria. Lactic acid bacteria can also be used for the production of food and pharmaceutical proteins, such as the production of sweet protein, which has the ability to secrete proteins to produce extracellular proteins and is considered to be a good protein expression system.

Many lactic acid bacteria expression systems have been described, and the systems currently used for heterologous protein expression can be roughly classified into continuous expression systems and inducible expression systems. The former can continue to trigger the expression of foreign genes; the latter needs to be in a specific environment to induce the expression of foreign genes.

A sustained performance system can consistently exhibit foreign proteins, but if it is harmful to the host, it can harm the host lactic acid bacteria and even cause death. The use of an inducible expression system avoids the damage caused by the continued expression of cytotoxic proteins.

The acid-induced expression system is a type of induction system whose promoter is induced by pH, which can metabolize acid production during growth and self-inducible to produce protein. However, self-induction does not facilitate control of the induction time, so that when the acid-induced expression system is to be applied, for example, to produce a sweet protein, it is difficult to induce production of a protein at an appropriate time point.

Accordingly, the present invention is directed to a method for producing a sweet protein using a controllable acid inducing system which can be induced at an appropriate time to produce a protein.

One aspect of the present invention is to provide a method of making a sweet protein using a controllable acid inducing system comprising the following steps. First, a recombinant plastid comprising an acid-inducible promoter and a deoxyribonucleic acid fragment as shown in SEQ ID NO: 2 is constructed. Next, the recombinant plasmid was introduced into Lactococcus lactic to obtain a transformed strain. Then, the transformed strain is cultured in a medium in which the pH of the medium is maintained at 6.5-7.5. When the transformant is grown at an optical density (OD ₆₀₀ ) value of 0.6-4.5 at a wavelength of 600 nm, 0.1 to 0.5 wt% of lactic acid is added to the medium to activate the acid-inducible promoter, thereby producing a DNA fragment. Sweet protein (brazzein).

According to an embodiment of the invention, wherein the acid-inducible promoter has a deoxyribonucleic acid fragment as shown in SEQ ID NO: 9.

According to an embodiment of the invention, wherein the medium is fermentation medium B (FMB).

According to an embodiment of the present invention, the content of lactic acid in the medium is from 0.1% by weight to 0.3% by weight to induce the production of protein by the lactic acid bacteria.

Therefore, by using the method, the amount of the transformed strain can be increased, and then induced at an appropriate time point (for example, appropriate cell density) to produce a large amount of sweet protein. Furthermore, the inducing agent of the method is lactic acid, which is not only cheap and easy to obtain, but also a food-grade reagent, so it is advantageous for the production of food or medical protein.

In the present invention, the amount of lactic acid bacteria is increased to a suitable cell density by using a medium, and then lactic acid is added to induce the acid-induced expression system of the lactic acid bacteria to produce a sweet protein.

The following will be divided into two parts. The first part explains the application basis of the embodiment of the present invention, and the second part illustrates the method of expressing the sweet taste protein by the acid-induced expression system in the lactic acid bacterium of the lactic acid.

first part:

The gene expression system of the P170 promoter is one of the acid-induced expression systems, which are induced by pH and use glucose as a carbon source. During the growth phase, the bacteria are converted from the postexponential phase to the stationary phase to produce acid, so that the pH of the environment is less than 6, thereby initiating self-induction to produce protein (hereinafter referred to as " Self-induced"). If the pH of the environment is greater than 6, the P170 promoter in the bacteria will not be activated.

In the protein part of the induced expression, brazzein is a sweet protein, which has the advantages of high sweetness, low calorie, good water solubility, good heat resistance and good pH stability as a sweetener. The taste is similar to that of sucrose and is not easily utilized by microorganisms. It does not cause oral cavities and is suitable for diabetic patients. It can be a substitute for nutritive carbohydrates in foods. Brazzein can now be purified from natural fruits, and the use of genetic engineering methods to produce brazzein sweet protein can make it more developed in the food industry. The sweet protein referred to below refers to brazzein.

The present invention utilizes a medium having a pH greater than 6, first increases the amount of lactic acid bacteria to a proper cell density, and then adds lactic acid to induce an acid-induced expression system of lactic acid bacteria, thereby controlling lactic acid bacteria to mass produce sweet protein (hereinafter referred to as "mandatory" Acid induction").

In the lactic acid bacteria induction test, a recombinant plastid that can be transduced with the sweet-sweet protein of sequence identification number 1 containing the gene sequence of the sweet-sweet protein (SEQ ID NO: 2) and the P170 promoter (sequence identification) is constructed. No. 3). This plastid was transformed into Lactococcus lacticus for subsequent self-induction tests and forced acid induction tests.

Please refer to Figure 1 for the self-induced test results of Streptococcus lacticus. The recombinant plastid-resistant nisin solution was inoculated overnight to a fresh fermentation medium B (FMB) in the form of 1.5% soy protein, 1% glucose. , 17 mM potassium dihydrogen phosphate (KH ₂ PO ₄ ), 72 mM dipotassium hydrogen phosphate (K ₂ HPO ₄ ), 1% yeast extract, 1 mM magnesium sulfate heptahydrate (MgSO 4‧7H ₂ O) And 0.1 mM manganese sulfate monohydrate (MnSO ₄ ‧ H ₂ O). The optical density (OD ₆₀₀ ) value of the initial wavelength of 600 nm was adjusted to 0.01, and the cultivation was continued. (A) is to measure the OD ₆₀₀ value of the culture for 2, 4, 6, 8, 10, 12 and 24 hours, (B) is to measure the pH value of the culture solution at the above time point, and (C) is to take the bacteria at the above time point. The solution was centrifuged to separate the cells and the supernatant, and a Western blot method was performed to detect the expression of the sweet protein.

Part I of Figure 1 (A) shows that the growth of lactic acid bacteria in OD ₆₀₀ tends to be flat at 4.5, and in the control (B) part, the pH of the P170 promoter is about 5.5-6.5. In the (C) part, the performance of the recombinant sweet protein was detected at the 10th hour.

Figure 2 shows the results of the forced acid induction test of Streptococcus lacticus. The recombinant plastid-containing S. mutans broth cultured overnight was inoculated to fresh FMB medium, and the initial OD ₆₀₀ value was adjusted to 0.01, and the culture was continued. Adding 0.1% by weight, 0.3% by weight, 0.5% by weight, 0.7% by weight or 1% by weight of lactic acid at 5 hours (about OD ₆₀₀ value of 0.673, pH 6.93), respectively, changing the pH to 6.67, 6.23, respectively. 5.57, 4.74, 4.12. (A) is to measure the OD ₆₀₀ absorbance at different time points; (B) to measure the pH of the culture solution at different time points; (C) to take the bacterial solution at the above time point, and centrifuge the isolated cells and the supernatant, Western blotting was performed to detect the performance of sweet protein.

Part of the results in Figure 2 (A) shows that the addition of 0.1% by weight and 0.3% by weight of lactic acid lactic acid bacteria continued to grow; (B) showed that the addition of lactic acid, the pH of the culture solution decreased faster than the group without lactic acid. In the (C) part, it was shown that the addition of 0.1% by weight of lactic acid could detect the expression of the recombinant sweet protein at the 8th hour, and the addition of 0.3% by weight of lactic acid could detect the expression of the recombinant sweet protein at the 10th hour. In contrast, when a group containing 0.5% by weight or more of lactic acid was added, the expression of the sweet protein could not be detected.

The second part:

In view of the above, one embodiment of the invention converts a segment of a DNA fragment encoding a sweet-sweet protein into S. lacticis. By adding lactic acid, the lactic acid bacteria are forced to induce acid production to produce a sweet protein.

A recombinant plastid having a coding-encoding heterologous protein suitable for use in the present invention, comprising an acid-inducible promoter, which is induced by pH to produce a heterologous protein. Unless otherwise specified, the "promoter" region described herein encompasses a promoter sequence recognized by an RNA polymerase, one or more regulatory sequences recognized by a regulatory gene, and induced phenotype initiation. Subsequence, transcription start point sequence.

Host lactic acid bacteria suitable for use in the present invention may include, but are not limited to, nisin. A suitable strain is a lactic acid strain having an acid-inducing system with appropriate regulatory factors and other essential elements for initiating an acid-inducing system.

Media suitable for use in the present invention include, but are not limited to, fermentation medium B. However, it should be noted that the fermentation medium B has the function of a buffer solution. Therefore, even if the number of bacteria increases, the pH does not change rapidly and the growth of the lactic acid bacteria is stagnant or even decreased, thereby increasing the bacterial cells. density.

In addition, according to the required culture and performance requirements, and different promoters have different induction conditions, the pH of the culture medium and the cell density to maintain the growth of the cells can be appropriately adjusted, and then induced at the appropriate time to achieve the desired The amount of protein expressed.

For example, according to the test results of the first part, the pH of the P170 promoter is between about 5.5 and 6.5. Therefore, in one example, the pH of the culture medium for the growth of the cells is maintained at 6.5-7.5. Increase the cell density. When the amount of the above-mentioned transformant strain reaches an optical density (OD ₆₀₀ ) value of 0.6 to 4.5 at a wavelength of 600 nm, lactic acid may be added to the medium to activate the acid-inducible promoter, thereby producing a sweet protein.

An embodiment of the present invention will be provided below to illustrate the method and effect of the present invention by expressing sweet protein.

Example: 1. Construction of the plastid (pNZABBA)

Figure 3 is a flow chart of the construction of the plastid (pNZABBA). In this example, the plastid pNZNSS with the levansucrase message peptide (SPsacB) and the sweet protein gene fragment of Babillus subtilis is used as a template, such as sequence identification number 4 And the primer pair shown in the sequence identification number 5 is subjected to gene amplification, wherein the obtained amplification product is a nucleic acid fragment represented by SEQ ID NO: 6, and the nucleic acid fragment represented by SEQ ID NO: 6 is designed to directly bind to the P170 promoter. Docked.

Then, the nucleic acid fragment shown by the sequence identification number 6 is used as a template, and the gene is amplified by using a primer pair as shown in the sequence identification number 7 and the sequence identification number 8 to obtain a nucleic acid fragment as shown in the sequence identification number 9, wherein The nucleic acid fragment represented by SEQ ID NO: 9 is an acid-inducible promoter-sweet protein expression deoxyribonucleic acid fragment of the expression cassette, which is referred to by the code ABBA.

The amplified fragment was first cut with Bgl II/ Xho I, and then ligated with the T4 ligase (T4 DNA ligase) at a suitable molar ratio by the vector pNZDSASm-SacBA2-4 which was cleaved by the same restriction enzyme. Completed pNZABBA plastid. The vector pNZDSASm-SacBA2-4 can be referred to "to enhance the secretion of the first type of recombinant antifreeze protein analogues in Streptococcus lacticis" (see Huang Xinhui, 2007), the promoter and transcription terminator from Lactobacillus acidophilus ; Lb. acidophilus ) Surface layer protein (Sslayer protein) (PslpA1, TslpA), message peptide (SPsacB) is derived from Bacillus subtilis fructan sucrase, screening marker is chloramphenicol Resistance gene.

The above plastid selection operation may include, but is not limited to, the following steps: the gene product amplified by the polymerase chain reaction may be first purified in a commercial kit, and the purified product may be subjected to a subsequent cleavage reaction.

The shearing system is carried out according to the procedure provided by the restriction enzyme manufacturer. In short, take appropriate amount of DNA sample, sterile deionized water, acetylated bovine serum albumin and restriction enzyme buffer into a microcentrifuge tube, add appropriate amount of restriction enzymes, mix well and place at the optimal reaction temperature of the enzyme. -4 hours, after the completion of the action, separation was carried out by agarose gel electrophoresis.

Thereafter, the colloid containing the target fragment is excised and recovered using a method of recovering DNA or a commercial kit commonly used in the art. The recovered product can be subjected to a bonding reaction.

The DNA ligation reaction is to take the vector DNA and the DNA fragment to be embedded, mix it in an appropriate ratio, add an appropriate amount of ligase buffer and sterilized deionized water to mix well, then add an appropriate amount of ligase to mix evenly, and react at a suitable reaction temperature of the enzyme. After the time, the reaction was terminated by heating at 65 ° C for 10 minutes. After the mixture was cooled, the transformation was carried out and transformed into JM109 competent cells of Escherichia coli.

Second, the transformation of E. coli

Transformation of the plastid into E. coli can be included but not limited to the following steps:

(1) Preparation of E. coli electric competent cells

The preparation of electrocompetent cells of E. coli was modified by the method published by Miller (1994). Pick a single colony and inoculate it in an appropriate amount of LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride (NaCl)), shake at 37 ° C, 150 rpm to cultivate. Inoculate one-hundredth of the volume of the overnight culture (12-16 hr) in fresh LB culture medium, shake culture at 37 ° C, 150 rpm until the OD ₆₀₀ value reaches 0.4-0.6, and the bacterial solution is ice-bathed for 30 minutes. Pour into a sterilized centrifuge bottle, centrifuge at 7000 x g for 15 minutes at 4 ° C, and discard the supernatant. The cells were washed three times with an equal volume of the original culture solution, half of the original culture solution volume, and 1 mL of ice-cold sterilized 10% glycerol (the cells were fully suspended, centrifuged at 7000 × g for 15 minutes at 4 ° C, and discarded. In addition to the supernatant). Finally, the cells were suspended in ice-cold sterilized 10% glycerol by one-fifth of the volume of the original culture solution, and were packed into several tubes (40 μL/tube), rapidly frozen in liquid nitrogen, and stored at -80 ° C.

(2) plastid transformation into Escherichia coli

Take 40 μL of the competent cells and melt on ice, add 1 μL of the plastid, and draw a mixture of competent cells and plastids, and place them in a pre-cooled cuvette. After 5 minutes in the ice bath, the electric field was transformed at an electric field strength of 2.25 kV, a resistance of 200 T, and a capacitance of 25 μF. The cells were regenerated in 1 mL of SB medium (35 g/L trypsin, 20 g/L yeast extract, 5 g/L sodium chloride), and cultured at 37 ° C and 120 rpm for 1 hour with shaking. After centrifugation at 10,000 × g for 10 minutes, the excess culture solution was removed, and then applied to an LB medium containing an appropriate amount of antibiotic, and cultured at 37 ° C for 16 hours.

(3) Screening of transformed plants

The transgenic strain was screened using colony polymerase chain reaction (colony PCR). This method uses a specific primer to perform a polymerase chain reaction on the target DNA to detect whether the colony contains the target DNA fragment. Add the dNTPs, primer pair, Gen TaqTM DNA polymerase (GeneMark Technology Co., Ltd, Taipei, Taiwan), 10X Gen Taq buffer and sterile deionized water to the total volume of the reaction in a 1.5 mL microcentrifuge tube, mix well and then dispense PCR tubules (10 μL/tube). The picked colonies were spotted from the medium with a toothpick to the PCR tubules for PCR reaction. The reaction conditions were: reaction at 95 ° C for 3 or 15 minutes (3 minutes for Escherichia coli; 15 minutes for subsequent screening of transformed strains of Streptococcus mutans), followed by 25 cycles of 95 ° C reaction for 30 seconds, primer adhesion temperature (Tm) The reaction was carried out for 30 seconds at 72 ° C for polymerase reaction (reaction time depending on the size of the amplified fragment, Gen TaqTM DNA polymerase: 1 kb/min), and further reacted at 72 ° C for 7 minutes. The product of the polymerase chain reaction was observed by agarose gel electrophoresis for the presence or absence of a DNA fragment of an estimated size.

(4) Confirmation of recombinant plastids

The transformed plastids confirmed by the colony polymerase chain reaction were extracted using a commercial kit or by a conventional extraction method, and the fragment size was observed by restriction enzyme cleavage to confirm that the target DNA fragment was indeed embedded. The plastids with the confirmed fragment size were subjected to DNA sequencing by Tri-I Biotech, Inc. (Taipei, Taiwan).

Third, the plastid is transformed into lactic acid bacteria

The transformation of the pNZABBA plastid into a lactic acid bacterium can be included but not limited to the following steps:

(1) Preparation of lactic acid bacteria competent cells:

The preparation method was modified according to the method published by Holo and Ness (1995). A single colony was picked and inoculated into GM17 culture solution (purchased from Difco, sterilized and then added with 0.5% lactose), and allowed to stand overnight at 30 ° C (12-16 hours), and then inoculated with one percent of the volume of the bacterial solution. 1% glycine (Glycine) fresh SGM17 medium (addition of 0.5M sucrose to GM17 medium), static culture at 30 ° C until the absorbance of OD ₆₀₀ reaches 0.2-0.7, the bacterial liquid ice bath 30 After the minute, the mixture was centrifuged at 7000 × g at 4 ° C for 10 minutes, and the supernatant was discarded. Ice-cold sterilization buffer I (0.5M sucrose, 0.1% glycerol) and half of the original culture solution in an equal volume of ice-cold sterilizing buffer II (0.5M sucrose, 10% glycerol, 0.05M ii) Ethylenediaminetetraacetic acid (EDTA), half of the original culture medium, ice-cold sterilization buffer I and 1 mL of ice-cold sterilization buffer I clean the cells 4 times (suspension of the cells by centrifugal force 7000 × g, 4 Centrifuge at °C for 15 minutes and remove the supernatant), resuspend in milliliters to 5% of the volume of the original culture medium in ice-cold sterilizing buffer I and dispense into several tubes (40 μL). Stored at -80 ° C after freezing.

(2) plastid transformation:

Take 40 μL of the competent cells and melt on ice. Add 1 μL of pNZABBA plastid to be transformed, and mix the competent cells and plastids into a pre-cooled cuvette. After 5 minutes on ice bath, The electric field was transformed at an electric field strength of 2.25 kV, a resistance of 200 T, and a capacitance of 25 μF. The cells were regenerated in 1 mL of SGM17MC medium (20 mM magnesium chloride (pH 6.8) and 2 mM calcium chloride (pH 6.8) were added to the SGM17 medium, and cultured at 30 ° C for 3 hours. Centrifugal force was centrifuged at 10,000 × g and applied to SR medium (1% tryptone, 0.5% yeast extract, 20% sucrose, 1% glucose, 2.5% gelatin, 1.5% agar) containing appropriate antibiotics. The cells were anaerobic cultured at 30 ° C for 16 hours.

4. Acid-induced system for expressing recombinant sweet protein in a nisin host

The NZ9000 with the pNZABBA plastid was cultured in the broth of the fermentation medium B containing the appropriate amount of antibiotics, and after standing overnight at 30 ° C, the broth was inoculated into a fresh fermentation medium B containing no antibiotics. In the solution, the pH of the culture medium in which the cells are grown is maintained at 6.5-7.5 to increase the cell density. Adding 0.1% by weight to 0.5% by weight of lactic acid at the 5th hour, continuing to grow to the 8th hour, centrifuging at 10000×g, 4° C. for 15 minutes, collecting the supernatant, and filtering the supernatant to remove the cells and A concentrated liquid is obtained from the acid molecule, and the concentrated ink is subjected to Western blotting to detect the expression of the sweet protein.

The recombinant plastid construction, the transformation effect and the screening of the transformed strain of the present invention can be easily accomplished according to the existing knowledge and technology in the technical field, and therefore will not be described here.

The present invention has been disclosed in the above embodiments, and is not intended to limit the present invention. Any one of ordinary skill in the art to which the present invention pertains can make various changes without departing from the spirit and scope of the invention. The scope of protection of the present invention is therefore defined by the scope of the appended claims.

<110>National Chung Hsing University

<120> Method for producing sweet protein using a controllable acid induction system

<160>9

<210>SEQ ID NO: 1

<211>

<212>PRT

<213>

<220>

<223>Amino acid sequence of brazzein

<400>1

<210>SEQ ID NO: 2

<211>165

<212>DNA

<213>

<220>CDS

<223> Gene sequence of sweet protein (brazzein)

<400>2

<210>SEQ ID NO: 3

<211>57

<212>DNA

<213>

<220>promoter

<223>P170 promoter

<400>3

<210>SEQ ID NO: 4

<211>89

<212>DNA

<213>Artificial sequence

<220>primer

<223> Performing PCR amplification sequence identification number 6 segment of the forward (forward)

<400>4

<210>SEQ ID NO: 5

<211>86

<212>DNA

<213>Artificial sequence

<220>primer

<223> Performing a PCR amplification sequence identification number 6 segment of the primer (reversed)

<400>5

<210>SEQ ID NO: 6

<211>373

<212>DNA

<213>Artificial sequence

<220>

<223>

<400>6

<210>SEQ ID NO: 7

<211>69

<212>DNA

<213>Artificial sequence

<220>primer

<223> Performing PCR amplification sequence identification number 9 segment of the forward (forward)

<400>7

<210>SEQ ID NO: 8

<211>29

<212>DNA

<213>Artificial sequence

<220>primer

<223> Performing a PCR amplification sequence identification number 9 fragment of the reversed

<400>8

<210>SEQ ID NO: 9

<211>412

<212>DNA

<213>Artificial sequence

<220>

<223> Acid-inducible promoter-sweet protein expression

<400>9

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

Figure 1 shows the results of self-induced testing of Streptococcus lacticus.

Figure 2 shows the results of the forced acid induction test of Streptococcus lacticus.

Figure 3 is a flow chart of the construction of the plastid (pNZABBA).

Claims (2)

A method for producing a sweet protein using a controllable acid-inducing system, comprising: constructing a recombinant plastid comprising an acid-inducible promoter and a DNA fragment as shown in SEQ ID NO: 2; introducing the recombinant plastid Lactococcus lactic acid to obtain a transformed strain; the transformed strain is cultured in fermentation medium B (FMB), wherein the pH of the fermentation medium B is maintained at 6.5-7.5; When the optical density (OD ₆₀₀ ) value of one of the transformants is 0.6-4.5 at a wavelength of 600 nm, 0.1% by weight to 0.3% by weight of lactic acid is added to the medium to activate the acid-inducing promoter, thereby The deoxyribonucleic acid fragment produces the sweet protein (brazzein). A method of producing a sweet protein using a controllable acid-inducing system according to claim 1, wherein the acid-inducing promoter has a deoxyribonucleic acid fragment as shown in SEQ ID NO: 9.