TW201231667A - Method for producing brazzein by using controllably acid-inducible system - Google Patents

Abstract

A method of producing brazzein by using the controllably acid-inducible system is disclosed. A recombinant plasmid that comprises an acid-inducible promoter and a polynucleotide encoding brazzein is introduced into Lactococcus lactis. The culture medium is adjusted to control the pH value of 6.5 to 7.5. When the growth of bacteria reaches OD600 of 0.6 to 4.5, lactic acid is added to induce the production of brazzein.

Description

201231667 VI. Description of the Invention: [Technical Field] The present invention relates to a method for producing a sweet protein, and more particularly to a method for producing a sweet protein using a controllable acid-inducing system. [Prior Art] Lactic acid bacteria are a group of quite complex bacteria. • Widely distributed in nature, they 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 is one of the most widely used and economically effective industrial bacteria. Lactic acid bacteria can also be used for the production of foods and pharmaceutical proteins, for example, the production of sweet protein, and the ability of lactic acid bacteria to secrete proteins to produce extracellular proteins, which is considered to be a good protein expression system. Many lactic acid bacteria expression systems have been described, and current systems for heterologous protein expression can be roughly classified into continuous performance systems and inducible expression systems. The former can continue to trigger the expression of foreign genes; the latter needs to induce foreign gene expression in a specific environment. Continuous 蜇 performance systems can consistently represent foreign proteins, but if they are harmful to the host, they can harm the host lactic acid bacteria and even cause death. The use of an inducible expression system 4 avoids the damage caused by the continued expression of cytotoxic proteins. The acid-induced expression system is a kind of induction system. The sputum promoter is induced by pH, and can metabolize acid production during growth. (10) (6) Stealing (6) 201231667 Protein production. However, the time required for self-induction is long, and it is not convenient to control 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. SUMMARY OF THE INVENTION 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, this recombinant plasmid was introduced into Streptococcus mutans (10) 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 to 7.5. When the transformant grows at an optical density (OD6G()) value of 0.6-4.5 at a wavelength of 600 nm, 0.1% by weight to 0.5% by weight of lactic acid is added to the medium to activate the acid-inducible promoter, thereby deoxyribonucleic acid The fragment produces a 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: 1. 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. 201231667 Therefore, 'this method can be used to increase the amount of transformed strains, and then induce at a time point (for example, appropriate g body density) to produce a large amount of sweet glutinous protein. Furthermore, the inducer of the method is lactic acid, which is not only cheap. It is easy to obtain, and it is a food-grade reagent, which is beneficial to the production of food or pharmaceutical protein. [Embodiment] / The present invention is to increase the amount of lactic acid (tetra) to an appropriate bacterial body and then add lactic acid to induce the acid of lactic acid bacteria to induce the production of sweet protein. The brothers and sisters will be divided into two parts. The first part will explain the application basis of the implementation of the present invention. The second part will explain the method of expressing the sweet egg self-quality in the drug-induced expression system in the larvae. Part I: The gene expression system of the Ρ170 promoter is a species of acid-induced expression system. This natural promoter is induced by ρΗ value and uses glucose as a carbon source. During the growth phase, the bacteria are converted from the post-exponential phase (p〇steXp〇nential 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-induction". If the pH of the environment is greater than 6, the pi7〇 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. Its taste is similar to that of strict sugar and is used by microorganisms. It does not cause oral tooth decay and is suitable for diabetic patients. 201231667 Use > 叮 to become a substitute for nutritive carbohydrates in food. Brazzein is purified from natural fruits and genetically engineered to produce bi*azzein astringent protein, which enables it to be further developed in the food industry. The sweet protein is brazzein. ^Inventive system uses a medium with a pH greater than 6, first extracts the amount of lactic acid bacteria to the appropriate cell density, and then adds lactic acid to induce the acid-induced lactic acid bacteria = expression system' to control the lactic acid bacteria to mass produce sweet protein (hereinafter referred to as "Compulsory acid induction"). In the acid-fast 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 number 3). This plastid was transformed into L. lactis for subsequent self-induction test and forced acid induction test. Please refer to Figure 1 for the results of the self-induced test of Streptococcus lacticus. The recombinant plastid-resistant S. mutans solution was inoculated overnight to a fresh fermentation medium B (FMB) formulated as 1.5% soy protein (SOyt〇ne), ι〇/〇. Glucose (giuc〇se), 17 mM dihydrogen hydride (ΚΗ2Ρ04), 72 mM acid hydrogen di-(κ2ΗΡ04) '1% yeast extract, 1 mM magnesium sulfate heptahydrate (MgS〇4. I wish 2〇) and o.imM — manganese sulfate (M11SO4 · H20). The optical density (OD_) value of the initial wavelength of 600 nm was adjusted to 〇.〇1, and the cultivation was continued. (a) to measure the d_ value after 2, 4, 6, 8, 10, 12, and 24 hours of culture, (B) to measure the pH of the culture solution at the above time point '(C) is at the above time point Take the halogen 'centrifugation of the cells and supernatant' to carry out the Western blot method to detect the performance of the sweet protein. Part (A) of the results in Figure 1 shows that 'the growth of Streptococcus mutans on 〇D(10) 7 201231667 tends to be flat when the absorbance reaches 4.5. In the control (B) part, the pH of the P170 promoter is about 5.5- Between 6.5, the performance of recombinant sweet protein was detected in the 10th hour in part (C). Figure 2 shows the results of the forced acid induction test of Streptococcus lacticus. The lactic acid-producing bacterium of the bacterium having a recombinant plastid cultured overnight was inoculated to fresh FMB medium, and the initial OD6 enthalpy 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_value of 0.673, pH 6.93), respectively, changing the pH to 6.67, 6.23, respectively. 5.57, 4.74, 4.12. (A) to measure the absorbance of 〇D6〇〇 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 supernatant Liquid, Western blotting method 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 performance 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. Part II: As stated above, one embodiment of the invention converts a segment of a DNA fragment encoding a sweet-sweet protein into S. lacticis. Borrowing 201231667, lactic acid is added to induce the lactic acid bacteria to induce the production of sweet protein. Applicable to the present invention is a coded-producing heterologous protein j body which comprises an acid-inducible promoter, is transduced by pH, and is of a quality. Unless otherwise specified, "= protein ΓΝΑ p°lymerase" as described herein: start: 表现 phenotype promoter sequence, transcription start point sequence. 砀 适用 适用 适用 适用 适用 适用 适用 _ _ _ _ _ _ Recorded in =. Applicable strains are lactic acid strains with an acid-inducing system, with the elements of the appropriate factor and other starting (four) guiding systems. The medium suitable for the present invention includes but is not limited to the fermentation medium to be described It is the function of the buffer of the fermentation medium, so (4) in the medium of his knowing that 'even if the rise' is not rapid and rapid, the growth of the lactic acid bacteria will be stagnant or even decreased, thus increasing the density of the bacteria. According to the required culture and performance requirements, and different promoters have different induction conditions, the pH value and the bacterial body temperature of the culture medium for maintaining the growth of the cells can be appropriately adjusted, and then induced at the appropriate time to achieve the desired The amount of protein expression. For example, according to the test results of the first part, the induced pH of the ρ17() promoter is between about 5·5_6 5 , so, in an example The pH of the culture medium for the growth of the cells is maintained at 6.5 7.5 to increase the density of the cells. When the amount of the above-mentioned transformed strain is at an optical density (〇d6gq) at a wavelength of 6 〇〇 nm, the value reaches 0.6-4.5. Lactic acid may be added to the medium to activate the acid-inducible promoter to produce a sweet protein. An embodiment of the present invention will now be provided to express the sweet protein to 201231667 to illustrate the method and effect of the present invention. The construction of the body (pNZABBA) is a flow chart of the plastid (pNZABBA). In this example, the 'flavinucrase' message with Bacillus subtilis &//& The peptide (SPsacB) and the plastid pNZNSS of the sweet protein gene fragment are used as templates, and the gene amplification is carried out by using a primer pair as shown in SEQ ID NO: 4 and sequence identification φ No. 5, wherein the obtained amplification product is, for example, a sequence identification number. The nucleic acid fragment shown in Figure 6, and the nucleic acid fragment shown in SEQ ID NO: 6 is designed to directly interface with the P170 promoter. Next, the nucleic acid fragment shown by SEQ ID NO: 6 is used as a template, such as sequence identification number 7 and The primer pair shown in Figure 8 recognizes the amplified gene to obtain a nucleic acid fragment as shown in SEQ ID NO: 9, wherein the nucleic acid fragment represented by SEQ ID NO: 9 is an acid-inducible promoter-sweet protein expression cassette (expression) The deoxyribonucleic acid fragment of cassette is called the code ABB A. The amplified fragment is first cut, and then the T4 ligase is used with the vector pNZDSASm-SacBA2-4 which is cleaved by the same restriction enzymes (T4 DNA ligase) The bonding was carried out at a suitable molar ratio to obtain a constructed pNZABBA plastid. The vector pNZDSASm-SacBA2-4 can be referred to "Improving the Secretory Expression of the First Type Recombinant Antifreeze Protein Analogs in Streptococcus Mutans" (see Huang Xinhui, 2007), whose promoter and transcription terminator are from LactobacUlus. Acidophilus', Lb. acidophiius) Surface layer protein; S-layer protein 201231667 (PslpAl, TslpA) 'Message peptide (spsacB) is derived from Bacillus subtilis fructanase Resistance gene of chloramphenicol. 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 cleaved and the method or quotient for recovering DN A commonly used in the art is utilized. The oral kit is recycled. The recovered product can be subjected to a bonding reaction. The DNA ligation reaction is to take the carrier DNA and the fragment to be embedded, mix it in an appropriate ratio, add an appropriate amount of ligase buffer and sterilized deionized water to mix evenly, and then add an appropriate amount of ligase to mix and knead, and react at a suitable reaction temperature of the enzyme. After the time, the reaction was terminated by heating at 65 ° C for 1 Torr. After the mixture was cooled, the transformation was carried out and transformed into JM109 competent cells of Escherichia coli. 2. Transformation of Escherichia coli Transformation of plastid into Escherichia coli can be included but not limited by the following steps: (1) Preparation of E. coli electric competent cells 201231667 E. coli electric competent cells (electr〇competent cell) The preparation was modified by the method published by Miller (1994). A single colony was picked and inoculated into an appropriate amount of LB medium (1% tryptone, 0.5% yeast extract, 1% sodium chloride (NaCl)) at 37 ° C, 150 rpm. Under the shock culture. Inoculate one-hundredth of the volume of the overnight culture (12-16 hr) in fresh LB medium, shake at 37 ° C, 150 rpm until the OD^oo value reaches 〇.4-〇·6, the bacterial solution Ice bath for 30 minutes 'pour into a sterilized centrifuge bottle' by centrifugation at 7 〇〇〇 xg, centrifuge at 4 ° C for 15 minutes, discard the supernatant. φ Wash the cells three times with an equal volume of the original culture solution, half the volume of the original culture solution, and 1 mL of ice-cold sterilized 10% glycerol (the cells were fully suspended, centrifuged at 7000 xg for 15 minutes at 4 ° C and Discard the supernatant). Finally, the cells were suspended by ice-cold sterilization of 10% glycerol in the original culture medium volume, and packed into several tubes (40 μί/tube), frozen in liquid nitrogen, and stored in _80〇c. (1) The plastid is transformed into E. coli and 40 pL of electric competent cells are thawed on ice, and a mixture of competent cells and plastids is taken in by adding 1 φ body, and placed into a pre-cooled electrode tube (cuvette). in. After 5 minutes of ice bathing, electrical transformation was performed at an electric field strength of 2.25 kV, a resistance of 200 T, and a capacitance of 25 pF. The cells were regenerated in 1 mL of SB medium (35 g/L trypsin, 20 g/L yeast extract, 5 g/L sodium), and cultured at 37 C, 120 rpm for 1 hour. Centrifuge at loooo Xg for 10 minutes to remove excess culture solution and apply to LB medium containing appropriate antibiotics at 37. (: Culture for 16 hours. (C) Screening of transgenic plants using colony polymerase chain reaction (colony PCR) screening (screening) 201231667 Transformed strain. This method uses a specific primer to polymerize the target DNA. Chain reaction to detect whether the colony contains the target DNA fragment. Add dNTPs, primer pair, Gen TaqTM DNA polymerase (GeneMark Technology Co., Ltd 5 Taipei, Taiwan), 1 〇x to a 1.5 mL microcentrifuge tube. The total volume of the reaction was adjusted by Gen Taq buffer and sterilized deionized water. After mixing, the mixture was dispensed into a PCR tube (10 μΐ^/tube). The picked colonies were picked up from the medium by a toothpick to the PCR tube for PCR reaction. The conditions were: reaction at 95 ° C for 3 or 15 minutes (3 minutes for Escherichia coli; 15 minutes for subsequent screening of the transformed strain of Streptococcus faecalis), followed by 25 cycles of 95 ° C reaction for 30 seconds, primer adhesion temperature ( Tm) reaction for 30 seconds, 72 ° C polymerase reaction (reaction time depends on the size of the fragment,

Gen TaqTM DNA polymerase: 1 kb/min), then react 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. (IV) Confirmation of recombinant plastids The plastids confirmed by colony polymerase chain reaction were extracted by commercial kits or by conventional extraction methods, and the fragment size was observed by restriction enzyme cleavage to confirm that it was indeed embedded. Target DNA fragment. The deacetylated nucleic acid sequencing was performed by Tri-I Biotech, Inc. (Taipei, Taiwan). 3. Transformation of plastids into lactic acid bacteria The transformation of pNZABBA plastids into lactic acid bacteria can be included but not limited by the following steps: (1) Preparation of lactic acid bacteria competent cells: 201231667 Preparation method refers to Holo and Ness (1995) The method was modified. A single colony was picked and inoculated into GM17 medium (purchased from Difco, sterilized and then added with 0.5% lactose) at 30. (: Allow overnight culture (12-16 hours), then inoculate one percent of the volume of the bacterial solution to fresh SGM17 medium containing 1% glycine (glyCine) (add 0.5V to the GM17 medium) In the sugar), the solution was allowed to stand at 30 ° C until the absorbance of OD_ reached 0.2-0.7, and the bacterial solution was ice-bathed for 3 minutes, then centrifuged at 7000 x g for 10 minutes at 4 ° C, and the supernatant was discarded. Iso-sterilized rinse buffer I (0 5 M sucrose, 0.1% glycerol) in the same volume as the original culture solution, and ice-cold sterilized rinse buffer II (0.5 M sucrose, 10% glycerol, 0.05 M ii) in half the volume of the original culture solution. Ethylenediaminetetraacetic acid (EDTA), half of the original culture medium, ice-cold sterilized rinse buffer I and 1 mL of ice-cold sterilized rinse buffer for cleaning the corpus callosum 4 times (after suspension of the cells, the centrifugal force is 7〇〇〇Xg) 4. Centrifuge for 15 minutes and remove the supernatant), then resuspend and split into several tubes (4〇^l) with one thousandth to one-fifth of the volume of the original culture solution. It is quickly frozen in liquid nitrogen and stored in -8 (TC. (2) plastid transformation: take 40 吣The cells were thawed on ice, and 1 μΙ^ 转 欲 欲 ΝΖΑΒΒΑ , , , , , , , , 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜 胜25kv, resistance 200T, capacitance 25卟 under electric transformation. Regenerated and cultured in i mL SGMHMC medium (add 2〇_magnesium chloride (PH6.8) and 2 mM chlorination (PH6.8) to SGMn medium) Medium, 3 (rc was statically cultured for 3 hours, the cells were concentrated at a force of 10_xg_ and concentrated in an SR medium containing appropriate amounts of antibiotics (1% tryptone, 〇.5% yeast extract, 2%%)蔑14 201231667 Sugar, 1% grape vine, 2.5 〇 / ° gelatin (gelatin), 1.5 〇 / o agar), anaerobic culture for 16 hours at 30 ° C. Fourth, the acid-induced system expresses recombinant sweet protein The nisin host is cultured in the broth of the fermentation medium B containing the appropriate amount of antibiotics, and is inoculated into the broth of the fermentation medium B containing the appropriate amount of antibiotics. After culturing overnight, the bacterium is inoculated with antibiotics. In the fresh fermentation medium B culture solution, the pH of the culture medium for the growth of the cells is maintained at 6.5-7.5 to increase the bacterial cells. Density. Add 0.1% by weight to 5% by weight of lactic acid at the 5th hour, continue to grow to the 8th hour, centrifuge at 1000 μg for 10 minutes, collect the supernatant, and collect the supernatant. Membrane filtration removes the bacteria and acid molecules to obtain a concentrate, and the concentrate 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-described embodiments, and is not intended to limit the invention. Any one of ordinary skill in the art to which the invention pertains can make various changes without departing from the spirit and scope of the invention. And the scope of the present invention is defined by the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS In order to make the above and other objects, features, advantages and embodiments of the present invention more obvious, the description of the drawings is as follows: 15 201231667 Figure 1 shows the results of self-induction test 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). [Main component symbol description] (none)

16 201231667 Sequence Listing <110> National Chung Hsing University <120> Method for producing sweet glutinous 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>

Asp Lys Cys Lys Lys Val Tyr Glu Asn Tyr Pro Val Ser Lys Cys 15 10 15

Gin Leu Ala Asn Gin Cys Asn Tyr Asp Cys Lys Leu Asp Lys His 20 25 30

Ala Arg Ser Gly Glu Cys Phe Tyr Asp Glu Lys Arg Asn Leu Gin 35 40 45

Cys lie Cys Asp Tyr Cys Glu Tyr 50 <210> SEQ ID NO: 2 <211>165

<212>DNA <213>

< 220 > CDS <223 > gene sequence of sweet protein (brazzein) <400>2 60 120 gataaatgta aaaaagttta tgaaaattat cctgtttcaa aatgtcaact tgcaaatcaa tgtaattatg actgcaaact tgataagcat gcgcgttcag gagaatgttt ttatgatgaa aaacgtaatc ttcaatgtat ttgtgattat tgtgaatatt aataa 165 <210> ID NO: 3 <211>57 <212>DNA<213><220> promoter 201231667 <223>P170 promoter <400>3 atttttggtt gccatttgtt aacgctgnnn nnnnnnnnnn ntgctataat nnnnnng 57 <210> SEQ ID NO: 4 < 211 > 89 < 2I2 > DNA < 213 > Artificial Sequence < 220 > Primer < 223 > Carrying PCR Amplification Sequence Identification No. 6 Fragment of Forward <400>

Cctcctctcc ctagtgctat aatacatatg agagctccag acaaaggagg tataaccata 60 tgaacatcaa aaagtttgca aaacaagca 89 <210> SEQ ID NO: 5 <211>86 <212>DNA<213>Artificial sequence<220>primer<223> Recognized number 6 fragment (ltured) <400>5 tttatactcg agtctagaaa aaagacagag ctttcgctct gcctttttca aagcttctgc 60 agttattaat attcacaata atcaca 86 <210> SEQ ID NO: 6 <211>373 <212>DNA <213> artificial sequence< 220 > < 223 > < 400 > 6 cctcctctcc ctagtgctat aatacatatg agagctccag acaaaggagg tataaccata 60 tgaacatcaa aaagtttgca aaacaagcaa cagtattaac ctttactacc gcactgctgg 120 caggaggcgc aactcaagcg tttgcggata aatgtaaaaa agtttatgaa aattatcctg 180 2 201231667 tttcaaaatg tcaacttgca aatcaatgta attatgactg caaacttgat aagcatgcgc gttcaggaga atgtttttat gatgaaaaac gtaatcttca atgtatttgt gattattgtg aatattaata actgcagaag ctttgaaaaa Ggcagagcga aagctctgtc ttttttctag actcgagtat aaa 373 240 300 360 <210> SEQ ID NO: 7 <211>69 <212>D NA < 213 > artificial sequence < 220 >primer < 223 > performs PCR amplification sequence identification number 9 segment of the forward (forward)

<400>7 ttataaagat ctatttttgg ttgccatttg ttaacgctgc ctcctctccc tagtgctata atacatatg 69 60 <210> SEQ ID NO: 8 <211>29 <212>DNA <213> Artificial Sequence<220 > Primer <223> Repliced sequence identification number 9 fragment <400>8 tttatactcg agtctagaaa aaagacaga 29

<210> SEQ ID NO: 9 <211>412 <212>DNA<213>Artificial sequence<220><223> Acid-induced promoter_sweet protein expression cassette<400>9 ttataaagat ctatttttgg ttgccatttg ttaacgctgc ctcctctccc tagtgctata atacatatga gagctccaga caaaggaggt ataaccatat gaacatcaaa aagtttgcaa aacaagcaac agtattaacc tttactaccg cactgctggc aggaggcgca actcaagcgt ttgcggataa atgtaaaaaa gtttatgaaa attatcctgt ttcaaaatgt caacttgcaa atcaatgtaa ttatgactgc aaacttgata agcatgcgcg ttcaggagaa tgtttttatg atgaaaaacg taatcttcaa tgtatttgtg attattgtga atattaataa 60 120 180 240 300 360 3 412 201231667 tttgaaaaag gcagagcgaa agctctgtct ctgcagaagc tttttctaga ctcgagtata Aa

4

Claims (1)

201231667 VII. Scope of Application: 1. A method for producing sweet protein using a controllable acid-inducing system, comprising: constructing a recombinant plastid comprising an acid-inducible promoter and deoxyribose as shown in SEQ ID NO: 2 a nucleic acid fragment; introducing the recombinant plastid into a lactic acid bacterium (ZaciococciAS.) to obtain a transformed strain; φ cultivating the transformed strain in a medium, wherein the pH of the medium is maintained at 6.5 - 7.5, and when the transformation When the optical density (OD600) value of the strain is 0.6-4.5 at a wavelength of 600 nm, 0.1% by weight to 0.5% by weight of lactic acid is added to the medium to activate the acid-inducing promoter, thereby deoxyribose The nucleic acid fragment produces the sweet protein (brazzein) 2. The method of claim 1, wherein the acid-inducible promoter has a sequence as shown in sequence identification number 1. a DNA fragment. 3. A method for producing a sweet protein using a controllable acid induction system according to claim 1, wherein the medium Fermentation medium B (FMB) 4. A method for producing a sweet protein using a controllable acid-inducing system according to claim 1, wherein the content of the lactic acid in the medium is 0.1 f L 201231667 by weight Percentage to 0.3 weight percent.