CN116716233A - Genetically engineered bacterium for producing staurosporine and preparation method thereof - Google Patents

Genetically engineered bacterium for producing staurosporine and preparation method thereof Download PDF

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CN116716233A
CN116716233A CN202310505030.7A CN202310505030A CN116716233A CN 116716233 A CN116716233 A CN 116716233A CN 202310505030 A CN202310505030 A CN 202310505030A CN 116716233 A CN116716233 A CN 116716233A
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star
staurosporine
genetically engineered
kaso
pset152
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黄浩龙
胡海峰
张梦莹
夏瑜泽
刘佳乐
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Sinopharm Health Industry Institute Co ltd
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Abstract

The invention discloses a genetic engineering bacterium for producing staurosporine, wherein the genetic engineering bacterium for producing staurosporine is micro Bai Lunci bacteria SIPI-ST-H-23 (Lentzea albida) and is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 27109; namely, engineering bacteria integrating the regulatory gene Star of the kasO p promoter in the genome of the original strain SIPI-ST-07 by utilizing an electrotransformation method. The fermentation yield of the genetically engineered bacterium is greatly improved, and the staurosporine yield is 1002mg/L. The micro Bai Lunci bacillus genetic engineering bacteria constructed by the invention can be directly used for the industrial production of midostaurin intermediate staurosporine, and can improve the yield and reduce the cost.

Description

Genetically engineered bacterium for producing staurosporine and preparation method thereof
Technical Field
The invention relates to the field of bioengineering, in particular to a genetically engineered bacterium for producing staurosporine, and a construction/preparation method and application thereof.
Background
Malignant tumors are diseases that severely threaten human health. Lung cancer, liver cancer, tumor of upper digestive system, colorectal cancer, male prostate cancer, female breast cancer and the like are still main malignant tumors in China. The relative survival rate of malignant tumors in China is about 40.5% in 5 years at present, and is improved by about 10 percent compared with the malignant tumors in 10 years ago, but the malignant tumors have a great gap with developed countries. Surgery, radiation therapy, chemotherapy and molecular targeted drugs remain several major approaches to the treatment of cancer, and therefore, more efficient antitumor drugs have been found to be the most effective approach to reduce mortality from malignant tumors. Midostaurin (trade name Rydapt), mainly obtained by a method of chemical synthesis from staurosporine, as shown in fig. 1; is a new drug for newly diagnosed FLT3 positive Acute Myelogenous Leukemia (AML) by being marketed by North US FDA approved by the United states of America in 4 of 2017 in combination with chemotherapy, and is also the first major breakthrough in leukemia treatment for 25 years. Rydapt acts as an oral multi-targeted kinase inhibitor, primarily by blocking several enzymes that promote cell growth, including FLT3, and has therefore been developed for the treatment of AML patients carrying FLT3 mutations.
FIG. 1 synthesis of midostaurin
Staurosporine is a synthetic precursor of midostaurin and has a molecular formula of C 28 H 26 N 4 O 3 Midostaurin is an antitumor drug, and at present, a large-scale microbial fermentation method is a main mode for producing staurosporine, but the problems of low yield and high production cost are probably one of the reasons that the current price of the midostaurin is high.
In the prior art, although the staurosporine production method disclosed in the patent CN101397540B can improve the staurosporine yield to a certain extent, the staurosporine yield is still lower, so that the new staurosporine production fermentation strain is constructed, the staurosporine yield is improved, the production cost is reduced, the operation steps are simplified, the environmental pollution is reduced, the yield is improved, and the requirement of industrial production is met.
Disclosure of Invention
The invention aims to solve the technical problem of lower yield in the fermentation process of staurosporine in the prior art, and provides a high-yield genetic engineering bacterium for producing staurosporine, a construction method and application thereof, which can improve the efficiency of producing staurosporine by using genetic engineering technology and reduce the cost.
With the development of molecular biology technology, the antibiotic biosynthesis gene cluster can be directionally transformed or modified by utilizing engineering means to obtain novel antibiotics or improve the yield of the antibiotics. However, the difficulty of the present invention is that the gene regulation mechanism of the staurosporine target product in its producer is not yet clear. According to the invention, through many researches and experiments, the control gene StaR is introduced into a staurosporine-producing host bacterium to enable the staurosporine to be over-expressed, and the content of staurosporine in the obtained engineering bacterium product is improved.
Therefore, one of the technical schemes of the invention is that a genetically engineered bacterium for producing staurosporine is characterized in that a regulatory gene StaR and a promoter ermE p or kasO p are integrated in the genome of a starting strain micro Bai Lunci (Lentzea albida) for producing staurosporine.
Preferably, the regulatory gene Star is derived from a self gene, and the promoter ermE p or kasO p is derived from an artificially synthesized exogenous gene fragment.
Preferably, the nucleotide sequence of the regulatory gene Star is shown as SEQ ID No. l, the nucleotide sequence of the promoter ermE p is shown as SEQ ID No.2, and the nucleotide sequence of the promoter kasO p is shown as SEQ ID No. 3.
The genetically engineered bacterium is micro Bai Lunci bacterium SIPI-ST-H-23 (Lentzea albida), and is preserved in China general microbiological culture Collection center (CGMCC) No.27109, and the preservation date is 2023, 4 and 13 days.
The invention further provides a technical scheme that: the preparation method of the genetically engineered bacterium comprises the following steps:
1) PCR artificially synthesizes whole genes of promoters ermE p and kasO p;
2) PCR amplification or artificial synthesis of the whole gene of Star;
3) Constructing a homologous recombination pSET152-StaR plasmid containing the StaR fragment, and transferring the plasmid into escherichia coli ET12567;
4) Constructing regulatory genes StaR recombinant pSET152-StaR-ermE p and pSET152-StaR-kasO p plasmids of different promoters ermE p and kasO p;
5) Electrotransformation of the constructed recombinant plasmid into a host bacterium SIPI-ST-07;
6) Electrotransformation and culture of new host bacteria with recombinant plasmid and produced staurosporine starter;
the nucleotide sequence of the plasmid pSET152-StaR is shown as SEQ ID No.4, the nucleotide sequence of the plasmid pSET152-StaR-ermE p is shown as SEQ ID No.5, and the nucleotide sequence of the plasmid pSET152-StaR-kasO p is shown as SEQ ID No. 6.
Preferably, the backbone pSET152 plasmid of the recombinant plasmid: preferably, the intermediate host bacterium is escherichia coli ET12567; preferably, the electrotransformation is performed in ISP2 agar medium at 28 ℃.
The other technical scheme of the invention is as follows: a method for preparing staurosporine comprises fermenting any one of the above genetically engineered bacteria to obtain staurosporine from fermentation broth. The culture and fermentation method can be selected from the existing culture and fermentation methods of staurosporine producing bacteria, especially micro Bai Lunci.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention. The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that: the invention adopts different promoters ermE p and kasO p to start the regulating gene StaR in the micro Bai Lunci bacteria of the staurosporine production bacteria, thereby improving the staurosporine yield, improving the production efficiency, reducing the production cost and reducing the environmental pollution.
Drawings
Fig. 1: pSET 152-Star-ermE.times.p plasmid map;
fig. 2: pSET152-Star-kasO p plasmid map;
fig. 3: HPLC profile of fermentation product of SIPI-ST-H-23 strain.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
The micro Bai Lunci strain SIPI-ST-07 from Lentzea albida was mutagenized to a staurosporine-producing high-yielding strain from this laboratory.
pSET152-SARP and pSBI153 plasmids are from the institute of plant physiology and ecology, national academy of sciences.
Glucose was purchased from national pharmaceutical group chemical reagent company, soybean cake powder was purchased from Qingdao Kerui biotechnology company, corn steep liquor dry powder was purchased from Shandong Si Wang Tangye Co., yeast powder was purchased from Angel Yeast Co., ltd, and the multi-segment recombination kit was purchased from Nannuo-NYO Biotech Co., ltd.
Example 1
(1) Amplification, cloning and expression of regulatory gene Star
Genome extraction of micro Bai Lunci (Lentzea albida) strain SIPI-ST-07 and amplification of regulatory Gene Star: SIPI-ST-07 was inoculated into 30mL of TSB medium (tryptone 1.5%, soytone 0.5%, naCl 0.5%, pH 7.2), and cultured at 28℃under shaking at 220rpm for 48 hours. The culture broth was centrifuged at 12000rpm for 5min, and the supernatant was collected to recover the cells. Genome extraction was performed using a bacterial genome DNA rapid extraction kit according to the extraction method provided by the supplier (bioengineering limited).
The working concentration of the primer is 50 mu M/L, and the working concentration of dNTP is 2.5mM/L. The PCR conditions were as follows: PCR amplification was performed using the primers StaR-R/F as primers and the SIPI-ST-07 genome as a template to obtain about 2700bp regulated gene StaR, and the reaction system (50. Mu.L) comprised 25. Mu.L of 2 XPCR buffer, 2. Mu.L of 2mM dNTPs, 1. Mu.L of FX enzyme, 2.5. Mu.L of DMSO, 17.5. Mu. L H 2 0, 1. Mu.L of each of the upstream and downstream primers, and 0.3. Mu.L of the template genome. The PCR reaction was performed at 95℃for 13 cycles (95℃30s,60℃30s,72℃90 s), 24 cycles (95℃30s,71℃30s,72℃90 s) for 10 minutes at 72℃and 16℃for 5 minutes. Specifically amplifying a DNA fragment of about 2700bp, subjecting the PCR product to agarose gel electrophoresis (Shanghai Techno Co., ltd.) at a voltage of 120V for 45min, performing gel recovery using a SanPrep column type DNA gel recovery kit (Biotechnology Co., ltd.), and finally dissolving in 30. Mu.L of pure water to obtain a StaR target fragment.
(2) Construction of expression plasmid pSET 152-Star:
the pSET152 plasmid was digested with BamHI and XbaI, the desired fragment pSET152 linear vector was recovered in a gel, and the pSET152 linear vector and the fragment of StaR were ligated using a multi-fragment recombination kit to obtain the expression plasmid pSET152-StaR.
(3) Construction of genetically engineered bacteria
Inoculating host bacteria SIPI-ST-07 to a fresh inclined plane, culturing for 5-6 d at 28 ℃, taking about 1cm multiplied by 1cm of thallus out by an inoculating shovel, inoculating the thallus into 30mLTSB liquid culture medium, culturing for 2d at 28 ℃ and 220rpm, transferring into 30mL TSB culture medium according to 1% inoculum size, culturing for 1d at 28 ℃ and 220 rpm. Centrifuging to remove the supernatant to obtain mycelium, washing with LB liquid medium for 2 times, and finally suspending in 2mL of LB liquid medium, which is the new host bacterial liquid.
Intermediate host E.coli ET12567 carrying plasmid pSET152-StaR was inoculated into 5mL LB liquid medium containing chloramphenicol, kanamycin, and apramycin at concentrations of 25. Mu.g/mL, 50. Mu.g/mL, and 50. Mu.g/mL, respectively, and cultured overnight at 37℃and 220 rpm. Transfer to 30mL of the same medium at 1% inoculum size, incubate at 37℃and 220rpm for about 24 hours, and then adjust the OD 600 The value is 1.0-1.2. The culture solution is used forThe mixture was centrifuged at 12000rpm for 5 minutes, and the supernatant was collected to recover the cells. Genome extraction was performed using a bacterial genome DNA rapid extraction kit according to the extraction method provided by the supplier (bioengineering limited). This is a fragment of the recombinant plasmid with the regulatory gene Star.
The host bacterial liquid and the recombinant plasmid fragment prepared above are added into an electrorotating cup according to the volume ratio of 1:1, the electric field strength is 12kV/cm, the resistance is 200 omega, the current is 25 mu F, the electrotransformation is carried out, and after full incubation, ISP2 culture medium is coated. Culturing at 28deg.C for 7d to obtain apramycin resistant strain, which is named SIPI-ST-A-01.
(4) Verification of genetically engineered bacteria
And (3) carrying out resistance verification on the apramycin resistant strain obtained in the step (3), respectively inoculating host bacteria and the obtained apramycin resistant genetic engineering strain to an inclined plane containing apramycin (50 mug/mL), and culturing for 6d at 28 ℃, wherein the host bacteria are observed not to grow on the inclined plane containing the apramycin, and the genetic engineering strain with the transformant can grow.
(5) Fermentation of genetically engineered bacteria
The composition of the plate medium was as follows: yeast extract 0.4%, malt extract 1%, glucose 0.4%, agar 2%, pH7.0, and culturing at 28deg.C for 7d.
The seed culture medium comprises the following components: glucose 2%, corn starch 0.5%, yeast powder 0.5%, corn steep liquor dry powder 0.5%, soybean peptone 0.5%, calcium carbonate 0.2% and pH 7.0. Inoculating the obtained genetically engineered bacteria into a seed culture medium, scraping a flat agar block with the length of 1cm multiplied by 2cm, inoculating the flat agar block into 100/750mL of the seed culture medium, and culturing at 28 ℃ for 48 hours to obtain a seed culture solution.
The composition of the fermentation tank (5L) culture medium comprises 4% of glucose, 1% of corn starch, 1.7% of cottonseed meal, 1.5% of soybean meal, 0.5% of yeast powder, 0.1% of monoammonium phosphate, 0.1% of cupric sulfate pentahydrate, 0.1% of magnesium sulfate heptahydrate, 0.05% of ferrous sulfate pentahydrate, 0.4% of calcium carbonate and 6.3 of pH value. Setting the culture temperature at 28 ℃, setting the air flow rate at 6L/min, setting the tank pressure at 0.03-0.05 MPa, stirring at 200-500rpm, fermenting for 5 days, and maintaining the dissolved oxygen in the fermentation tank at more than 20%.
TABLE 1 primer sequences for plasmid construction
Example 2
(1) Amplification, cloning and expression of regulatory gene Star
The corresponding Star gene fragment was obtained according to the method in example 1.
(2) Amplification, cloning and expression of promoter ermE.times.p
Coli ET12567 containing pSET152-SARP was inoculated into apramycin (50. Mu.g/mL) resistant solid LB, cultured overnight at 37℃and single colonies were picked up and transferred to 4mL apramycin (50. Mu.g/mL) resistant liquid LB, and plasmids were extracted after shaking overnight at 37 ℃. The reaction system (50. Mu.L) comprises 25. Mu.L of 2 XPCR buffer, 2. Mu.L of 2mM dNTPs, 1. Mu.LFX enzyme, 2.5. Mu.L of DMSO, 17.5. Mu. L H 2 0, 1. Mu.L of each of the upstream and downstream primers, and 0.3. Mu.L of the template genome. The PCR reaction was performed at 95℃for 13 cycles (95℃30s,60℃30s,72℃90 s), 24 cycles (95℃30s,71℃30s,72℃90 s) for 10 minutes at 72℃and 16℃for 5 minutes. The gel recovered about 220bp promoter ermE.times.p.
(3) Construction of expression plasmid pSET 152-Star-ermE. Times.p:
the pSET152-StaR plasmid is subjected to double digestion reaction with BamHI and XbaI, a target fragment pSET152-StaR linear vector is recovered by glue, and the target fragment pSET152-StaR linear vector is respectively connected with a promoter ermE p fragment by using a multi-fragment recombination kit to obtain an expression plasmid pSET152-StaR-ermE p.
(4) Construction of genetically engineered bacteria
The construction was carried out in the same manner as in (3) in example 1. The genetically engineered bacterium is named SIPI-ST-G-5.
(5) Verification of genetically engineered bacteria
Resistance verification was performed on the apramycin-resistant strain obtained in the above step (4), and the method was the same as in the step (4) of example 1.
(6) Fermentation of genetically engineered bacteria
And (3) fermenting the genetically engineered bacteria obtained in the step (5) respectively, wherein the method is the same as that in the step (5) of the example 1.
TABLE 2 primer sequences for plasmid construction
Example 3
(1) Amplification, cloning and expression of regulatory gene Star
The corresponding Star gene fragment was obtained according to the method in example 1.
(2) Amplification, cloning and expression of the promoter kasO p
Coli ET12567 containing pSBI153 was inoculated into apramycin (50. Mu.g/mL) resistant solid LB, cultured overnight at 37℃and single colonies were picked up and transferred to 4mL apramycin (50. Mu.g/mL) resistant liquid LB, and plasmids were extracted after shaking overnight culture at 37 ℃.
The pSBI153 plasmid was digested with XbaI/EcoRI restriction enzyme at 37℃for 2 to 3 hours, and the promoter kasO p of about 100bp was recovered on gel. The reaction system (50. Mu.L) comprises 25. Mu.L of 2 XPCR buffer, 2. Mu.L of 2mM dNTPs, 1. Mu.L of FX enzyme, 2.5. Mu.L of DMSO, 17.5. Mu. L H 2 0, 1. Mu.L of each of the upstream and downstream primers, and 0.3. Mu.L of the template genome. The PCR reaction was performed at 95℃for 13 cycles (95℃30s,60℃30s,72℃90 s), 24 cycles (95℃30s,71℃30s,72℃90 s) for 10 minutes at 72℃and 16℃for 5 minutes. The gel recovered about 100bp promoter kasO p.
(3) Construction of expression plasmid pSET 152-Star-kasO. Times.p:
the pSET152-Star plasmid was digested with BamH1 and Xba1, the desired fragment pSET152-Star linear vector was recovered by gel ligation with the promoter kasO p fragment using a multi-fragment recombination kit to obtain the expression plasmid pSET152-Star-kasO p.
(4) Construction of genetically engineered bacteria
The construction was carried out in the same manner as in (3) in example 1. The genetically engineered bacterium is named as SIPI-ST-H-23.
(5) Verification of genetically engineered bacteria
Resistance verification was performed on the apramycin-resistant strain obtained in the above step (4), and the method was the same as in the step (4) of example 1.
(6) Fermentation of genetically engineered bacteria
And (3) fermenting the genetically engineered bacteria obtained in the step (5) respectively, wherein the method is the same as that in the step (5) of the example 1.
TABLE 3 primer sequences for plasmid construction
Example 4
Analysis of staurosporine-producing strain fermentation products: the recombinant plasmid-containing genetically engineered bacteria obtained in examples 1 to 3 and the wild SIPI-ST-07 host bacteria without recombinant plasmid were fermented, and then analyzed for staurosporine content by HPLC. The HPLC analysis method is briefly described as follows: the fermentation broth is pretreated, 2mL of culture for 7d of drunk culture is added with 4mL of 95% ethanol and mixed uniformly, 250W ultrasonic treatment is carried out for 10min,2504 Xg is centrifugated for 10min, supernatant is taken, and the supernatant is filtered by a 0.22 mu m filter membrane and then is used for HIPLC method measurement. Chromatographic conditions: chromatographic column NovaC18-4μm/>(Waters) column: mobile phase a (acetonitrile): mobile phase B (0.02 mol/L sodium acetate solution); mobile phase ratio a: b=46: 54 isocratic elution; the flow rate is 1.0mL/min; column temperature is 35 ℃; detection wavelength 292nm; the sample injection amount was 10. Mu.L. The staurosporine content of the fermentation broth was calculated based on the concentration of the standard and the results are shown in table 1 below:
TABLE 4 fermentation products of genetically engineered bacteria
Name of the name SIPI-ST-07 SIPI-ST-A-01 SIPI-ST-G-5 SIPI-ST-H-23
Yield (mg/L) 845 836 899 1002
From the above examples it can be concluded that the genetically engineered bacteria integrating the regulatory gene Star and the promoter kasO p have the most pronounced effect on the product. The staurosporine yield of the SIPI-ST-H-23 genetically engineered bacteria reaches 1002mg/L, which is improved by 1.3 times compared with the original strain.
TABLE 5 list of sequences
Numbering device Name of the name Purpose(s)
SEQ ID No.1 StaR Regulatory genes
SEQ ID No.2 erm*E Promoters
SEQ ID No.3 KasO*p Promoters
SEQ ID No.4 pSET152-StaR Homologous recombination plasmid
SEQ ID No.5 pSET152-StaR-ermE*p Homologous recombinant plasmid of erythromycin promoter
SEQ ID No.6 pSET152-StaR-kasO*p Homologous recombinant plasmid with strong promoter
Nucleotide sequence table SEQ ID No.1 of regulatory gene Star:
atgagggagaaaatgcacagctgttcacggtcggcaatggcaaagtgcgagaatccggttctcaacgcagttcacacacagcccatttcccggggagcagtgcacggcagggaagaccaactggcccagacgatcacgcagctgcgcgccgtggcgagcagcgggcggagcacggtgctggtgctgggggcggttcccggcgccggcaagacccggctcctcagggaggcggcgtcgctcgccgagagcgacggcttcatggtgctgagcgccgccccggccgggcccgacaccacccccgaggccgtcatcgaggcggcgcggacacggctccccggcgccgggagcacccgcaccaccgtcgtcctcgacgatctgcatctggcgggactccccgcactgacgacgctgaacgacctcgtcgtggccctcaggggacgtccgatcctctggctgctcgccttcaccacggaacgcgacgccccacccccggaacacctgcgggcctctctcggcggactccgcgccagcctcccggtggagcacatgcgggagctgggaccgatcgagggcgacgcgctggcacagttggtcgccgaccacaccggagccacccccgacccggctctcctcgccctcgcggagagcgtcaacgccacgccgcgctcggtgatcgagttgattcgcggcctggcggaggacgacgacctgtgcctgatcgacggcaccacccgattacagcccggcccacccggcgacccctcggccgggtggggcgtcccggtgcccacccccgtaccgaaacggttctccgcgaccgtccagcaagacctcaggacactgtcggaccccaccctgaaggcgctcagactggccgccgtcctcggatcaccgttcgcaccggaagacctgtcggccctgctcgacgaagcacccgtcggtctgctcgccgccgtggacgaggccgtcgaccggggcctgctcgtctgcggcgaacgcgacctcgccttccgcaccgagccgatctggcgggtcctgctcgactccgtaccgcccccggtgctcgccctgctgcgccggcaggccgcgaagatcgtgctgccgcgccccgacggcgtcgagcgcgccgccctccaactggtccacgtcgcccaacccggcaacacggacgaactgcgcatcatcgccgagggctcccgcagactcctgctcgccgacccgtccgcagccgcctcgctcgccacccgctccatggagttcctggaccccggcaccgccgagcgggtgcgactggcacggaccgccgtcgaagcgctcaccagagccggccgactggaccacgcgatcgccctggccaaggacaccgtcgacgagacagcccgcctcgccgccccactgccgccggagctcgtcgaggacgtcgccgcgttgcgggcctcgatgtcgaccgccctgctgctcctcggcgacgcccgcctcgcccgccgagcggcaggcgacgcgctcgcggtacagcacggcgggccccagcaccgcgaggcggtcgtcgcccacctcgccgcctcctacctcaccggcgacgccagcgccgcccagcgcgcacgccagatcctcggcgcacccgaccgccacaccggcgccgtgcaggtgggcgcgatgaccttccacgccttcggccagtggcgcgccggcgacgtcggcgaggccgtggccaccctgcgcagggcggtcgccctcgaccgcgccgacgacgaggcgccgatcctcgacccccgctggttcctcaccttcaccctcgccaggaccgacgagtacgagcaggcggcggccgtcatccggagctccgccgccatcgcctcgaaacacggcacgctgaccaccgcggtccccgccgtgctccgcgcccaactgaacctcgcacagggccggttggacgaggcggaggacgacgccaagctcggggtcggcaccgacggcccctttgtgccgatgctcgccccgcaggcatggctggttctagcctccgtggccctgcgccgaggagcgctcgcccaggccgaggaacacatcaagaccctggagaaggacttcccccagcacgcctccagcccctggtggggcgcgcggctgctgctcaacgcgcagttggccgaggcacaggccgaccagcacgccgcgacggaggtgctggccgagatcggggcgcagaacggtgcgctccgcgaggtcgtcctggaggatccggccgcggcggcctggtgggtacggtgcgctctcgcggccgagcgacccgacctcgtctcgacggtgatcgaggcgacggagcacctccgtggacgcaaccagtgcgtgccctcggtggtggccatggccatgcacgcccgggcgctcgccgaaggggacgccgaggcactcgaccgggccgggcggctacaccgcaatccctgggcgcaggcgaccgcggccgaggaccacgcggggctgctcctcgaccgtggggagcacgaggccgccatcggtgagttcgaccgggcgatgagcgccttcggcgcgctcggcggggagcgggacgccgcccgggtgcgggcgcggctgcgtgcgctgggggtacggcggcggcactggacgcatgcgaaacgtccggtgtccgggtgggagagcctgaccaagaccgagcggaaggtggccgagctggtggccggggggctcaccaatcagcaggccgcccggcatctgttcatctcaccgcacacggtcgggttccatctgcgccagatctaccggaagttgggcatccggtcgcgcaccgcgctgattcggctgagggcgtga
nucleotide sequence table SEQ ID No.2 of promoter erme×p:
catgcgagtgtccgttcgagtggcggcttgcgcccgatgctagtcgcggttgatcggcgatcgcaggtgcacgcggtcgatcttgacggctggcgagaggtgcggggaggatctgaccgacgcggtccggttggtaggatccacat
nucleotide sequence table of promoter kasO p SEQ ID No.3:
catgcgagcgacgcggtccacacacgtggcaccgcgatgctgttgtgggcacaatcgtgccggttggtaggatccact
nucleotide sequence table SEQ ID No.4 of plasmid pSET 152-Star:
atctacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtctccgacctgatgcagctctcgcagggcgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtttctacaaagatcgttatgttgatcggcactttgcatcggccgcgctcccgattccggaagtgcttgacattggggaatttatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccaagcttgggctgcaggtcgactctagaggatccgcggccgcgcgcgatatcgaattcgtaatcatatgagggagaaaatgcacagctgttcacggtcggcaatggcaaagtgcgagaatccggttctcaacgcagttcacacacagcccatttcccggggagcagtgcacggcagggaagaccaactggcccagacgatcacgcagctgcgcgccgtggcgagcagcgggcggagcacggtgctggtgctgggggcggttcccggcgccggcaagacccggctcctcagggaggcggcgtcgctcgccgagagcgacggcttcatggtgctgagcgccgccccggccgggcccgacaccacccccgaggccgtcatcgaggcggcgcggacacggctccccggcgccgggagcacccgcaccaccgtcgtcctcgacgatctgcatctggcgggactccccgcactgacgacgctgaacgacctcgtcgtggccctcaggggacgtccgatcctctggctgctcgccttcaccacggaacgcgacgccccacccccggaacacctgcgggcctctctcggcggactccgcgccagcctcccggtggagcacatgcgggagctgggaccgatcgagggcgacgcgctggcacagttggtcgccgaccacaccggagccacccccgacccggctctcctcgccctcgcggagagcgtcaacgccacgccgcgctcggtgatcgagttgattcgcggcctggcggaggacgacgacctgtgcctgatcgacggcaccacccgattacagcccggcccacccggcgacccctcggccgggtggggcgtcccggtgcccacccccgtaccgaaacggttctccgcgaccgtccagcaagacctcaggacactgtcggaccccaccctgaaggcgctcagactggccgccgtcctcggatcaccgttcgcaccggaagacctgtcggccctgctcgacgaagcacccgtcggtctgctcgccgccgtggacgaggccgtcgaccggggcctgctcgtctgcggcgaacgcgacctcgccttccgcaccgagccgatctggcgggtcctgctcgactccgtaccgcccccggtgctcgccctgctgcgccggcaggccgcgaagatcgtgctgccgcgccccgacggcgtcgagcgcgccgccctccaactggtccacgtcgcccaacccggcaacacggacgaactgcgcatcatcgccgagggctcccgcagactcctgctcgccgacccgtccgcagccgcctcgctcgccacccgctccatggagttcctggaccccggcaccgccgagcgggtgcgactggcacggaccgccgtcgaagcgctcaccagagccggccgactggaccacgcgatcgccctggccaaggacaccgtcgacgagacagcccgcctcgccgccccactgccgccggagctcgtcgaggacgtcgccgcgttgcgggcctcgatgtcgaccgccctgctgctcctcggcgacgcccgcctcgcccgccgagcggcaggcgacgcgctcgcggtacagcacggcgggccccagcaccgcgaggcggtcgtcgcccacctcgccgcctcctacctcaccggcgacgccagcgccgcccagcgcgcacgccagatcctcggcgcacccgaccgccacaccggcgccgtgcaggtgggcgcgatgaccttccacgccttcggccagtggcgcgccggcgacgtcggcgaggccgtggccaccctgcgcagggcggtcgccctcgaccgcgccgacgacgaggcgccgatcctcgacccccgct
ggttcctcaccttcaccctcgccaggaccgacgagtacgagcaggcggcggccgtcatccggagctccgccgccatcgcctcgaaacacgg
cacgctgaccaccgcggtccccgccgtgctccgcgcccaactgaacctcgcacagggccggttggacgaggcggaggacgacgccaagct
cggggtcggcaccgacggcccctttgtgccgatgctcgccccgcaggcatggctggttctagcctccgtggccctgcgccgaggagcgctcg
cccaggccgaggaacacatcaagaccctggagaaggacttcccccagcacgcctccagcccctggtggggcgcgcggctgctgctcaacg
cgcagttggccgaggcacaggccgaccagcacgccgcgacggaggtgctggccgagatcggggcgcagaacggtgcgctccgcgaggt
cgtcctggaggatccggccgcggcggcctggtgggtacggtgcgctctcgcggccgagcgacccgacctcgtctcgacggtgatcgaggcg
acggagcacctccgtggacgcaaccagtgcgtgccctcggtggtggccatggccatgcacgcccgggcgctcgccgaaggggacgccga
ggcactcgaccgggccgggcggctacaccgcaatccctgggcgcaggcgaccgcggccgaggaccacgcggggctgctcctcgaccgtg
gggagcacgaggccgccatcggtgagttcgaccgggcgatgagcgccttcggcgcgctcggcggggagcgggacgccgcccgggtgcg
ggcgcggctgcgtgcgctgggggtacggcggcggcactggacgcatgcgaaacgtccggtgtccgggtgggagagcctgaccaagaccg
agcggaaggtggccgagctggtggccggggggctcaccaatcagcaggccgcccggcatctgttcatctcaccgcacacggtcgggttccat
ctgcgccagatctaccggaagttgggcatccggtcgcgcaccgcgctgattcggctgagggcgtgagtcatagctgtttcctgtgtgaaattgtt
atccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttg
cgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggc
gctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatcca
cagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgttttt
ccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgt
ttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttct
catagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgc
cttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgagg
tatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagtta
ccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaa
aaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatca
aaaaggatcttcacctagatccttttggttcatgtgcagctccatcagcaaaaggggatgataagtttatcaccaccgactatttgcaacagtgccgt
tgatcgtgctatgatcgactgatgtcatcagcggtggagtgcaatgtcgtgcaatacgaatggcgaaaagccgagctcatcggtcagcttctcaa
ccttggggttacccccggcggtgtgctgctggtccacagctccttccgtagcgtccggcccctcgaagatgggccacttggactgatcgaggcc
ctgcgtgctgcgctgggtccgggagggacgctcgtcatgccctcgtggtcaggtctggacgacgagccgttcgatcctgccacgtcgcccgtt
acaccggaccttggagttgtctctgacacattctggcgcctgccaaatgtaaagcgcagcgcccatccatttgcctttgcggcagcggggccac
aggcagagcagatcatctctgatccattgcccctgccacctcactcgcctgcaagcccggtcgcccgtgtccatgaactcgatgggcaggtactt
ctcctcggcgtgggacacgatgccaacacgacgctgcatcttgccgagttgatggcaaaggttccctatggggtgccgagacactgcaccattc
ttcaggatggcaagttggtacgcgtcgattatctcgagaatgaccactgctgtgagcgctttgccttggcggacaggtggctcaaggagaagag
ccttcagaaggaaggtccagtcggtcatgcctttgctcggttgatccgctcccgcgacattgtggcgacagccctgggtcaactgggccgagat
ccgttgatcttcctgcatccgccagaggcgggatgcgaagaatgcgatgccgctcgccagtcgattggctgagctcatgagcggagaacgag
atgacgttggaggggcaaggtcgcgctgattgctggggcaacacgtggagcggatcggggattgtctttcttcagctcgctgatgatatgctga
cgctcaatgccgtttggcctccgactaacgaaaatcccgcatttggacggctgatccgattggcacggcggacggcgaatggcggagcagac
gctcgtccgggggcaatgagatatgaaaaagcctgaactcaccgcgacgtatcgggccctggccagctagctagagtcgacctgcaggtccc
cggggatcggtcttgccttgctcgtcggtgatgtacttcaccagctccgcgaagtcgctcttcttgatggagcgcatggggacgtgcttggcaatc
acgcgcaccccccggccgttttagcggctaaaaaagtcatggctctgccctcgggcggaccacgcccatcatgaccttgccaagctcgtcctg
cttctcttcgatcttcgccagcagggcgaggatcgtggcatcaccgaaccgcgccgtgcgcgggtcgtcggtgagccagagtttcagcaggcc
gcccaggcggcccaggtcgccattgatgcgggccagctcgcggacgtgctcatagtccacgacgcccgtgattttgtagccctggccgacgg
ccagcaggtaggccgacaggctcatgccggccgccgccgccttttcctcaatcgctcttcgttcgtctggaaggcagtacaccttgataggtgg
gctgcccttcctggttggcttggtttcatcagccatccgcttgccctcatctgttacgccggcggtagccggccagcctcgcagagcaggattccc
gttgagcaccgccaggtgcgaataagggacagtgaagaaggaacacccgctcgcgggtgggcctacttcacctatcctgcccggctgacgcc
gttggatacaccaaggaaagtctacacgaaccctttggcaaaatcctgtatatcgtgcgaaaaaggatggatataccgaaaaaatcgctataatg
accccgaagcagggttatgcagcggaaaagatccgtcgacctgcaggcatgcaagctctagcgattccagacgtcccgaaggcgtggcgcg
gcttccccgtgccggagcaatcgccctgggtgggttacacgacgcccctctatggcccgtactgacggacacaccgaagccccggcggcaa
ccctcagcggatgccccggggcttcacgttttcccaggtcagaagcggttttcgggagtagtgccccaactggggtaacctttgagttctctcagt
tgggggcgtagggtcgccgacatgacacaaggggttgtgaccggggtggacacgtacgcgggtgcttacgaccgtcagtcgcgcgagcgc
gagaattcgagcgcagcaagcccagcgacacagcgtagcgccaacgaagacaaggcggccgaccttcagcgcgaagtcgagcgcgacg
ggggccggttcaggttcgtcgggcatttcagcgaagcgccgggcacgtcggcgttcgggacggcggagcgcccggagttcgaacgcatcct
gaacgaatgccgcgccgggcggctcaacatgatcattgtctatgacgtgtcgcgcttctcgcgcctgaaggtcatggacgcgattccgattgtct
cggaattgctcgccctgggcgtgacgattgtttccactcaggaaggcgtcttccggcagggaaacgtcatggacctgattcacctgattatgcgg
ctcgacgcgtcgcacaaagaatcttcgctgaagtcggcgaagattctcgacacgaagaaccttcagcgcgaattgggcgggtacgtcggcgg
gaaggcgccttacggcttcgagcttgtttcggagacgaaggagatcacgcgcaacggccgaatggtcaatgtcgtcatcaacaagcttgcgca
ctcgaccactccccttaccggacccttcgagttcgagcccgacgtaatccggtggtggtggcgtgagatcaagacgcacaaacaccttcccttc
aagccgggcagtcaagccgccattcacccgggcagcatcacggggctttgtaagcgcatggacgctgacgccgtgccgacccggggcgag
acgattgggaagaagaccgcttcaagcgcctgggacccggcaaccgttatgcgaatccttcgggacccgcgtattgcgggcttcgccgctgag
gtgatctacaagaagaagccggacggcacgccgaccacgaagattgagggttaccgcattcagcgcgacccgatcacgctccggccggtcg
agcttgattgcggaccgatcatcgagcccgctgagtggtatgagcttcaggcgtggttggacggcagggggcgcggcaaggggctttcccgg
gggcaagccattctgtccgccatggacaagctgtactgcgagtgtggcgccgtcatgacttcgaagcgcggggaagaatcgatcaaggactct
taccgctgccgtcgccggaaggtggtcgacccgtccgcacctgggcagcacgaaggcacgtgcaacgtcagcatggcggcactcgacaagt
tcgttgcggaacgcatcttcaacaagatcaggcacgccgaaggcgacgaagagacgttggcgcttctgtgggaagccgcccgacgcttcggc
aagctcactgaggcgcctgagaagagcggcgaacgggcgaaccttgttgcggagcgcgccgacgccctgaacgcccttgaagagctgtac
gaagaccgcgcggcaggcgcgtacgacggacccgttggcaggaagcacttccggaagcaacaggcagcgctgacgctccggcagcaag
gggcggaagagcggcttgccgaacttgaagccgccgaagccccgaagcttccccttgaccaatggttccccgaagacgccgacgctgaccc
gaccggccctaagtcgtggtgggggcgcgcgtcagtagacgacaagcgcgtgttcgtcgggctcttcgtagacaagatcgttgtcacgaagtc
gactacgggcagggggcagggaacgcccatcgagaagcgcgcttcgatcacgtgggcgaagccgccgaccgacgacgacgaagacgac
gcccaggacggcacggaagacgtagcggcgtagcgagacacccgggaagcctg
nucleotide sequence table SEQ ID No.5 of plasmid pSET152-StaR-ermE p:
atctacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtctccgacctgatgcagctctcgcagggcgaagaatctcgtgctttcagcttc
gatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtttctacaaagatcgttatgttgatcggcactttgcatcggccgcg
ctcccgattccggaagtgcttgacattggggaatttatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattc
gccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggc
gattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccaagcttgggctgcaggtcgactctagaggatc
cgcggccgcgcgcgatatcgaattcgtaatcatcatgcgagtgtccgttcgagtggcggcttgcgcccgatgctagtcgcggttgatcggcgat
cgcaggtgcacgcggtcgatcttgacggctggcgagaggtgcggggaggatctgaccgacgcggtccggttggtaggatccacatatgagg
gagaaaatgcacagctgttcacggtcggcaatggcaaagtgcgagaatccggttctcaacgcagttcacacacagcccatttcccggggagca
gtgcacggcagggaagaccaactggcccagacgatcacgcagctgcgcgccgtggcgagcagcgggcggagcacggtgctggtgctggg
ggcggttcccggcgccggcaagacccggctcctcagggaggcggcgtcgctcgccgagagcgacggcttcatggtgctgagcgccgcccc
ggccgggcccgacaccacccccgaggccgtcatcgaggcggcgcggacacggctccccggcgccgggagcacccgcaccaccgtcgtc
ctcgacgatctgcatctggcgggactccccgcactgacgacgctgaacgacctcgtcgtggccctcaggggacgtccgatcctctggctgctc
gccttcaccacggaacgcgacgccccacccccggaacacctgcgggcctctctcggcggactccgcgccagcctcccggtggagcacatgc
gggagctgggaccgatcgagggcgacgcgctggcacagttggtcgccgaccacaccggagccacccccgacccggctctcctcgccctcg
cggagagcgtcaacgccacgccgcgctcggtgatcgagttgattcgcggcctggcggaggacgacgacctgtgcctgatcgacggcaccac
ccgattacagcccggcccacccggcgacccctcggccgggtggggcgtcccggtgcccacccccgtaccgaaacggttctccgcgaccgtc
cagcaagacctcaggacactgtcggaccccaccctgaaggcgctcagactggccgccgtcctcggatcaccgttcgcaccggaagacctgtc
ggccctgctcgacgaagcacccgtcggtctgctcgccgccgtggacgaggccgtcgaccggggcctgctcgtctgcggcgaacgcgacctc
gccttccgcaccgagccgatctggcgggtcctgctcgactccgtaccgcccccggtgctcgccctgctgcgccggcaggccgcgaagatcgt
gctgccgcgccccgacggcgtcgagcgcgccgccctccaactggtccacgtcgcccaacccggcaacacggacgaactgcgcatcatcgc
cgagggctcccgcagactcctgctcgccgacccgtccgcagccgcctcgctcgccacccgctccatggagttcctggaccccggcaccgcc
gagcgggtgcgactggcacggaccgccgtcgaagcgctcaccagagccggccgactggaccacgcgatcgccctggccaaggacaccgt
cgacgagacagcccgcctcgccgccccactgccgccggagctcgtcgaggacgtcgccgcgttgcgggcctcgatgtcgaccgccctgctg
ctcctcggcgacgcccgcctcgcccgccgagcggcaggcgacgcgctcgcggtacagcacggcgggccccagcaccgcgaggcggtcg
tcgcccacctcgccgcctcctacctcaccggcgacgccagcgccgcccagcgcgcacgccagatcctcggcgcacccgaccgccacaccg
gcgccgtgcaggtgggcgcgatgaccttccacgccttcggccagtggcgcgccggcgacgtcggcgaggccgtggccaccctgcgcagg
gcggtcgccctcgaccgcgccgacgacgaggcgccgatcctcgacccccgctggttcctcaccttcaccctcgccaggaccgacgagtacg
agcaggcggcggccgtcatccggagctccgccgccatcgcctcgaaacacggcacgctgaccaccgcggtccccgccgtgctccgcgccc
aactgaacctcgcacagggccggttggacgaggcggaggacgacgccaagctcggggtcggcaccgacggcccctttgtgccgatgctcg
ccccgcaggcatggctggttctagcctccgtggccctgcgccgaggagcgctcgcccaggccgaggaacacatcaagaccctggagaagg
acttcccccagcacgcctccagcccctggtggggcgcgcggctgctgctcaacgcgcagttggccgaggcacaggccgaccagcacgccg
cgacggaggtgctggccgagatcggggcgcagaacggtgcgctccgcgaggtcgtcctggaggatccggccgcggcggcctggtgggta
cggtgcgctctcgcggccgagcgacccgacctcgtctcgacggtgatcgaggcgacggagcacctccgtggacgcaaccagtgcgtgccct
cggtggtggccatggccatgcacgcccgggcgctcgccgaaggggacgccgaggcactcgaccgggccgggcggctacaccgcaatccc
tgggcgcaggcgaccgcggccgaggaccacgcggggctgctcctcgaccgtggggagcacgaggccgccatcggtgagttcgaccgggc
gatgagcgccttcggcgcgctcggcggggagcgggacgccgcccgggtgcgggcgcggctgcgtgcgctgggggtacggcggcggcac
tggacgcatgcgaaacgtccggtgtccgggtgggagagcctgaccaagaccgagcggaaggtggccgagctggtggccggggggctcac
caatcagcaggccgcccggcatctgttcatctcaccgcacacggtcgggttccatctgcgccagatctaccggaagttgggcatccggtcgcg
caccgcgctgattcggctgagggcgtgagtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagca
taaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgcc
agctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtc
gttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagc
aaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatc
gacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgac
cctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcg
ttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaaga
cacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggccta
actacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaac
aaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggg
gtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttggttcatgtgcagc
tccatcagcaaaaggggatgataagtttatcaccaccgactatttgcaacagtgccgttgatcgtgctatgatcgactgatgtcatcagcggtgga
gtgcaatgtcgtgcaatacgaatggcgaaaagccgagctcatcggtcagcttctcaaccttggggttacccccggcggtgtgctgctggtccac
agctccttccgtagcgtccggcccctcgaagatgggccacttggactgatcgaggccctgcgtgctgcgctgggtccgggagggacgctcgtc
atgccctcgtggtcaggtctggacgacgagccgttcgatcctgccacgtcgcccgttacaccggaccttggagttgtctctgacacattctggcg
cctgccaaatgtaaagcgcagcgcccatccatttgcctttgcggcagcggggccacaggcagagcagatcatctctgatccattgcccctgcca
cctcactcgcctgcaagcccggtcgcccgtgtccatgaactcgatgggcaggtacttctcctcggcgtgggacacgatgccaacacgacgctg
catcttgccgagttgatggcaaaggttccctatggggtgccgagacactgcaccattcttcaggatggcaagttggtacgcgtcgattatctcgag
aatgaccactgctgtgagcgctttgccttggcggacaggtggctcaaggagaagagccttcagaaggaaggtccagtcggtcatgcctttgctc
ggttgatccgctcccgcgacattgtggcgacagccctgggtcaactgggccgagatccgttgatcttcctgcatccgccagaggcgggatgcg
aagaatgcgatgccgctcgccagtcgattggctgagctcatgagcggagaacgagatgacgttggaggggcaaggtcgcgctgattgctggg
gcaacacgtggagcggatcggggattgtctttcttcagctcgctgatgatatgctgacgctcaatgccgtttggcctccgactaacgaaaatcccg
catttggacggctgatccgattggcacggcggacggcgaatggcggagcagacgctcgtccgggggcaatgagatatgaaaaagcctgaac
tcaccgcgacgtatcgggccctggccagctagctagagtcgacctgcaggtccccggggatcggtcttgccttgctcgtcggtgatgtacttcac
cagctccgcgaagtcgctcttcttgatggagcgcatggggacgtgcttggcaatcacgcgcaccccccggccgttttagcggctaaaaaagtca
tggctctgccctcgggcggaccacgcccatcatgaccttgccaagctcgtcctgcttctcttcgatcttcgccagcagggcgaggatcgtggcat
caccgaaccgcgccgtgcgcgggtcgtcggtgagccagagtttcagcaggccgcccaggcggcccaggtcgccattgatgcgggccagct
cgcggacgtgctcatagtccacgacgcccgtgattttgtagccctggccgacggccagcaggtaggccgacaggctcatgccggccgccgc
cgccttttcctcaatcgctcttcgttcgtctggaaggcagtacaccttgataggtgggctgcccttcctggttggcttggtttcatcagccatccgctt
gccctcatctgttacgccggcggtagccggccagcctcgcagagcaggattcccgttgagcaccgccaggtgcgaataagggacagtgaag
aaggaacacccgctcgcgggtgggcctacttcacctatcctgcccggctgacgccgttggatacaccaaggaaagtctacacgaaccctttgg
caaaatcctgtatatcgtgcgaaaaaggatggatataccgaaaaaatcgctataatgaccccgaagcagggttatgcagcggaaaagatccgtc
gacctgcaggcatgcaagctctagcgattccagacgtcccgaaggcgtggcgcggcttccccgtgccggagcaatcgccctgggtgggttac
acgacgcccctctatggcccgtactgacggacacaccgaagccccggcggcaaccctcagcggatgccccggggcttcacgttttcccaggt
cagaagcggttttcgggagtagtgccccaactggggtaacctttgagttctctcagttgggggcgtagggtcgccgacatgacacaaggggttg
tgaccggggtggacacgtacgcgggtgcttacgaccgtcagtcgcgcgagcgcgagaattcgagcgcagcaagcccagcgacacagcgta
gcgccaacgaagacaaggcggccgaccttcagcgcgaagtcgagcgcgacgggggccggttcaggttcgtcgggcatttcagcgaagcgc
cgggcacgtcggcgttcgggacggcggagcgcccggagttcgaacgcatcctgaacgaatgccgcgccgggcggctcaacatgatcattgt
ctatgacgtgtcgcgcttctcgcgcctgaaggtcatggacgcgattccgattgtctcggaattgctcgccctgggcgtgacgattgtttccactcag
gaaggcgtcttccggcagggaaacgtcatggacctgattcacctgattatgcggctcgacgcgtcgcacaaagaatcttcgctgaagtcggcga
agattctcgacacgaagaaccttcagcgcgaattgggcgggtacgtcggcgggaaggcgccttacggcttcgagcttgtttcggagacgaagg
agatcacgcgcaacggccgaatggtcaatgtcgtcatcaacaagcttgcgcactcgaccactccccttaccggacccttcgagttcgagcccga
cgtaatccggtggtggtggcgtgagatcaagacgcacaaacaccttcccttcaagccgggcagtcaagccgccattcacccgggcagcatca
cggggctttgtaagcgcatggacgctgacgccgtgccgacccggggcgagacgattgggaagaagaccgcttcaagcgcctgggacccgg
caaccgttatgcgaatccttcgggacccgcgtattgcgggcttcgccgctgaggtgatctacaagaagaagccggacggcacgccgaccacg
aagattgagggttaccgcattcagcgcgacccgatcacgctccggccggtcgagcttgattgcggaccgatcatcgagcccgctgagtggtat
gagcttcaggcgtggttggacggcagggggcgcggcaaggggctttcccgggggcaagccattctgtccgccatggacaagctgtactgcg
agtgtggcgccgtcatgacttcgaagcgcggggaagaatcgatcaaggactcttaccgctgccgtcgccggaaggtggtcgacccgtccgca
cctgggcagcacgaaggcacgtgcaacgtcagcatggcggcactcgacaagttcgttgcggaacgcatcttcaacaagatcaggcacgccg
aaggcgacgaagagacgttggcgcttctgtgggaagccgcccgacgcttcggcaagctcactgaggcgcctgagaagagcggcgaacggg
cgaaccttgttgcggagcgcgccgacgccctgaacgcccttgaagagctgtacgaagaccgcgcggcaggcgcgtacgacggacccgttg
gcaggaagcacttccggaagcaacaggcagcgctgacgctccggcagcaaggggcggaagagcggcttgccgaacttgaagccgccgaa
gccccgaagcttccccttgaccaatggttccccgaagacgccgacgctgacccgaccggccctaagtcgtggtgggggcgcgcgtcagtaga
cgacaagcgcgtgttcgtcgggctcttcgtagacaagatcgttgtcacgaagtcgactacgggcagggggcagggaacgcccatcgagaagc
gcgcttcgatcacgtgggcgaagccgccgaccgacgacgacgaagacgacgcccaggacggcacggaagacgtagcggcgtagcgaga
cacccgggaagcctg
nucleotide sequence table of plasmid pSET152-StaR-kasO p SEQ ID No.6:
atctacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtctccgacctgatgcagctctcgcagggcgaagaatctcgtgctttcagcttc
gatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtttctacaaagatcgttatgttgatcggcactttgcatcggccgcg
ctcccgattccggaagtgcttgacattggggaatttatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattc
gccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggc
gattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgccaagcttgggctgcaggtcgactctagaggatc
cgcggccgcgcgcgatatcgaattcgtaatcatcatgcgagcgacgcggtccacacacgtggcaccgcgatgctgttgtgggcacaatcgtgc
cggttggtaggatccactatgagggagaaaatgcacagctgttcacggtcggcaatggcaaagtgcgagaatccggttctcaacgcagttcaca
cacagcccatttcccggggagcagtgcacggcagggaagaccaactggcccagacgatcacgcagctgcgcgccgtggcgagcagcggg
cggagcacggtgctggtgctgggggcggttcccggcgccggcaagacccggctcctcagggaggcggcgtcgctcgccgagagcgacgg
cttcatggtgctgagcgccgccccggccgggcccgacaccacccccgaggccgtcatcgaggcggcgcggacacggctccccggcgccg
ggagcacccgcaccaccgtcgtcctcgacgatctgcatctggcgggactccccgcactgacgacgctgaacgacctcgtcgtggccctcagg
ggacgtccgatcctctggctgctcgccttcaccacggaacgcgacgccccacccccggaacacctgcgggcctctctcggcggactccgcgc
cagcctcccggtggagcacatgcgggagctgggaccgatcgagggcgacgcgctggcacagttggtcgccgaccacaccggagccaccc
ccgacccggctctcctcgccctcgcggagagcgtcaacgccacgccgcgctcggtgatcgagttgattcgcggcctggcggaggacgacga
cctgtgcctgatcgacggcaccacccgattacagcccggcccacccggcgacccctcggccgggtggggcgtcccggtgcccacccccgta
ccgaaacggttctccgcgaccgtccagcaagacctcaggacactgtcggaccccaccctgaaggcgctcagactggccgccgtcctcggatc
accgttcgcaccggaagacctgtcggccctgctcgacgaagcacccgtcggtctgctcgccgccgtggacgaggccgtcgaccggggcctg
ctcgtctgcggcgaacgcgacctcgccttccgcaccgagccgatctggcgggtcctgctcgactccgtaccgcccccggtgctcgccctgctg
cgccggcaggccgcgaagatcgtgctgccgcgccccgacggcgtcgagcgcgccgccctccaactggtccacgtcgcccaacccggcaa
cacggacgaactgcgcatcatcgccgagggctcccgcagactcctgctcgccgacccgtccgcagccgcctcgctcgccacccgctccatg
gagttcctggaccccggcaccgccgagcgggtgcgactggcacggaccgccgtcgaagcgctcaccagagccggccgactggaccacgc
gatcgccctggccaaggacaccgtcgacgagacagcccgcctcgccgccccactgccgccggagctcgtcgaggacgtcgccgcgttgcg
ggcctcgatgtcgaccgccctgctgctcctcggcgacgcccgcctcgcccgccgagcggcaggcgacgcgctcgcggtacagcacggcgg
gccccagcaccgcgaggcggtcgtcgcccacctcgccgcctcctacctcaccggcgacgccagcgccgcccagcgcgcacgccagatcct
cggcgcacccgaccgccacaccggcgccgtgcaggtgggcgcgatgaccttccacgccttcggccagtggcgcgccggcgacgtcggcg
aggccgtggccaccctgcgcagggcggtcgccctcgaccgcgccgacgacgaggcgccgatcctcgacccccgctggttcctcaccttcac
cctcgccaggaccgacgagtacgagcaggcggcggccgtcatccggagctccgccgccatcgcctcgaaacacggcacgctgaccaccgc
ggtccccgccgtgctccgcgcccaactgaacctcgcacagggccggttggacgaggcggaggacgacgccaagctcggggtcggcaccg
acggcccctttgtgccgatgctcgccccgcaggcatggctggttctagcctccgtggccctgcgccgaggagcgctcgcccaggccgaggaa
cacatcaagaccctggagaaggacttcccccagcacgcctccagcccctggtggggcgcgcggctgctgctcaacgcgcagttggccgagg
cacaggccgaccagcacgccgcgacggaggtgctggccgagatcggggcgcagaacggtgcgctccgcgaggtcgtcctggaggatccg
gccgcggcggcctggtgggtacggtgcgctctcgcggccgagcgacccgacctcgtctcgacggtgatcgaggcgacggagcacctccgt
ggacgcaaccagtgcgtgccctcggtggtggccatggccatgcacgcccgggcgctcgccgaaggggacgccgaggcactcgaccgggc
cgggcggctacaccgcaatccctgggcgcaggcgaccgcggccgaggaccacgcggggctgctcctcgaccgtggggagcacgaggcc
gccatcggtgagttcgaccgggcgatgagcgccttcggcgcgctcggcggggagcgggacgccgcccgggtgcgggcgcggctgcgtgc
gctgggggtacggcggcggcactggacgcatgcgaaacgtccggtgtccgggtgggagagcctgaccaagaccgagcggaaggtggccg
agctggtggccggggggctcaccaatcagcaggccgcccggcatctgttcatctcaccgcacacggtcgggttccatctgcgccagatctacc
ggaagttgggcatccggtcgcgcaccgcgctgattcggctgagggcgtgagtcatagctgtttcctgtgtgaaattgttatccgctcacaattcca
cacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgcttt
ccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcg
ctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggata
acgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccc
cctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctc
cctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgta
ggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatc
gtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgcta
cagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagag
ttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaag
aagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacc
tagatccttttggttcatgtgcagctccatcagcaaaaggggatgataagtttatcaccaccgactatttgcaacagtgccgttgatcgtgctatgat
cgactgatgtcatcagcggtggagtgcaatgtcgtgcaatacgaatggcgaaaagccgagctcatcggtcagcttctcaaccttggggttaccc
ccggcggtgtgctgctggtccacagctccttccgtagcgtccggcccctcgaagatgggccacttggactgatcgaggccctgcgtgctgcgct
gggtccgggagggacgctcgtcatgccctcgtggtcaggtctggacgacgagccgttcgatcctgccacgtcgcccgttacaccggaccttgg
agttgtctctgacacattctggcgcctgccaaatgtaaagcgcagcgcccatccatttgcctttgcggcagcggggccacaggcagagcagatc
atctctgatccattgcccctgccacctcactcgcctgcaagcccggtcgcccgtgtccatgaactcgatgggcaggtacttctcctcggcgtggg
acacgatgccaacacgacgctgcatcttgccgagttgatggcaaaggttccctatggggtgccgagacactgcaccattcttcaggatggcaag
ttggtacgcgtcgattatctcgagaatgaccactgctgtgagcgctttgccttggcggacaggtggctcaaggagaagagccttcagaaggaag
gtccagtcggtcatgcctttgctcggttgatccgctcccgcgacattgtggcgacagccctgggtcaactgggccgagatccgttgatcttcctgc
atccgccagaggcgggatgcgaagaatgcgatgccgctcgccagtcgattggctgagctcatgagcggagaacgagatgacgttggagggg
caaggtcgcgctgattgctggggcaacacgtggagcggatcggggattgtctttcttcagctcgctgatgatatgctgacgctcaatgccgtttgg
cctccgactaacgaaaatcccgcatttggacggctgatccgattggcacggcggacggcgaatggcggagcagacgctcgtccgggggcaa
tgagatatgaaaaagcctgaactcaccgcgacgtatcgggccctggccagctagctagagtcgacctgcaggtccccggggatcggtcttgcc
ttgctcgtcggtgatgtacttcaccagctccgcgaagtcgctcttcttgatggagcgcatggggacgtgcttggcaatcacgcgcaccccccggc
cgttttagcggctaaaaaagtcatggctctgccctcgggcggaccacgcccatcatgaccttgccaagctcgtcctgcttctcttcgatcttcgcca
gcagggcgaggatcgtggcatcaccgaaccgcgccgtgcgcgggtcgtcggtgagccagagtttcagcaggccgcccaggcggcccaggt
cgccattgatgcgggccagctcgcggacgtgctcatagtccacgacgcccgtgattttgtagccctggccgacggccagcaggtaggccgac
aggctcatgccggccgccgccgccttttcctcaatcgctcttcgttcgtctggaaggcagtacaccttgataggtgggctgcccttcctggttggct
tggtttcatcagccatccgcttgccctcatctgttacgccggcggtagccggccagcctcgcagagcaggattcccgttgagcaccgccaggtg
cgaataagggacagtgaagaaggaacacccgctcgcgggtgggcctacttcacctatcctgcccggctgacgccgttggatacaccaaggaa
agtctacacgaaccctttggcaaaatcctgtatatcgtgcgaaaaaggatggatataccgaaaaaatcgctataatgaccccgaagcagggttat
gcagcggaaaagatccgtcgacctgcaggcatgcaagctctagcgattccagacgtcccgaaggcgtggcgcggcttccccgtgccggagc
aatcgccctgggtgggttacacgacgcccctctatggcccgtactgacggacacaccgaagccccggcggcaaccctcagcggatgccccg
gggcttcacgttttcccaggtcagaagcggttttcgggagtagtgccccaactggggtaacctttgagttctctcagttgggggcgtagggtcgc
cgacatgacacaaggggttgtgaccggggtggacacgtacgcgggtgcttacgaccgtcagtcgcgcgagcgcgagaattcgagcgcagca
agcccagcgacacagcgtagcgccaacgaagacaaggcggccgaccttcagcgcgaagtcgagcgcgacgggggccggttcaggttcgt
cgggcatttcagcgaagcgccgggcacgtcggcgttcgggacggcggagcgcccggagttcgaacgcatcctgaacgaatgccgcgccgg
gcggctcaacatgatcattgtctatgacgtgtcgcgcttctcgcgcctgaaggtcatggacgcgattccgattgtctcggaattgctcgccctggg
cgtgacgattgtttccactcaggaaggcgtcttccggcagggaaacgtcatggacctgattcacctgattatgcggctcgacgcgtcgcacaaa
gaatcttcgctgaagtcggcgaagattctcgacacgaagaaccttcagcgcgaattgggcgggtacgtcggcgggaaggcgccttacggcttc
gagcttgtttcggagacgaaggagatcacgcgcaacggccgaatggtcaatgtcgtcatcaacaagcttgcgcactcgaccactccccttaccg
gacccttcgagttcgagcccgacgtaatccggtggtggtggcgtgagatcaagacgcacaaacaccttcccttcaagccgggcagtcaagccg
ccattcacccgggcagcatcacggggctttgtaagcgcatggacgctgacgccgtgccgacccggggcgagacgattgggaagaagaccg
cttcaagcgcctgggacccggcaaccgttatgcgaatccttcgggacccgcgtattgcgggcttcgccgctgaggtgatctacaagaagaagc
cggacggcacgccgaccacgaagattgagggttaccgcattcagcgcgacccgatcacgctccggccggtcgagcttgattgcggaccgatc
atcgagcccgctgagtggtatgagcttcaggcgtggttggacggcagggggcgcggcaaggggctttcccgggggcaagccattctgtccgc
catggacaagctgtactgcgagtgtggcgccgtcatgacttcgaagcgcggggaagaatcgatcaaggactcttaccgctgccgtcgccggaa
ggtggtcgacccgtccgcacctgggcagcacgaaggcacgtgcaacgtcagcatggcggcactcgacaagttcgttgcggaacgcatcttca
acaagatcaggcacgccgaaggcgacgaagagacgttggcgcttctgtgggaagccgcccgacgcttcggcaagctcactgaggcgcctga
gaagagcggcgaacgggcgaaccttgttgcggagcgcgccgacgccctgaacgcccttgaagagctgtacgaagaccgcgcggcaggcg
cgtacgacggacccgttggcaggaagcacttccggaagcaacaggcagcgctgacgctccggcagcaaggggcggaagagcggcttgcc
gaacttgaagccgccgaagccccgaagcttccccttgaccaatggttccccgaagacgccgacgctgacccgaccggccctaagtcgtggtg
ggggcgcgcgtcagtagacgacaagcgcgtgttcgtcgggctcttcgtagacaagatcgttgtcacgaagtcgactacgggcagggggcag
ggaacgcccatcgagaagcgcgcttcgatcacgtgggcgaagccgccgaccgacgacgacgaagacgacgcccaggacggcacggaag
acgtagcggcgtagcgagacacccgggaagcctg。

Claims (8)

1. a genetically engineered strain for producing staurosporine, characterized in that a regulatory gene StaR and a promoter ermE p or kasO p are integrated in the genome of a starting strain micro Bai Lunci (Lentzea albida) for producing staurosporine.
2. The genetically engineered bacterium of claim 1, wherein the regulatory genes StaR and promoter kasO x p are integrated into the genome of a statzea albida (inczea albida) starting strain producing staurosporine.
3. The genetically engineered bacterium of claim 1, wherein the regulatory gene Star is derived from a self gene and the promoter ermE.times.p or kasO.times.p is derived from an artificially synthesized foreign gene fragment.
4. The genetically engineered bacterium of claim 1, wherein the regulatory gene Star has a nucleotide sequence shown in SEQ ID No. l, the promoter ermE p has a nucleotide sequence shown in SEQ ID No.2, and the promoter kasO p has a nucleotide sequence shown in SEQ ID No. 3.
5. The genetically engineered bacterium of claim 1, wherein the genetically engineered bacterium is micro Bai Lunci bacteria SIPI-ST-H-23 (luntzea albida) deposited in China general microbiological culture collection center with a deposit number of CGMCC No.27109.
6. A method for producing the genetically engineered bacterium of any one of claims 1 to 5, comprising the steps of:
1) PCR artificially synthesizes whole genes of promoters ermE p and kasO p;
2) PCR amplification or artificial synthesis of the whole gene of Star;
3) Constructing a homologous recombination pSET152-StaR containing the StaR fragment, and transferring the fragment into escherichia coli ET12567;
4) Constructing regulatory genes StaR recombinant pSET152-StaR-ermE p and pSET152-StaR-kasO p plasmids of different promoters ermE p and kasO p;
5) Electrotransformation of the constructed recombinant plasmid into a host bacterium SIPI-ST-07;
6) Electrotransformation and culture of new host bacteria with recombinant plasmid and produced staurosporine starter;
the nucleotide sequence of the plasmid pSET152-StaR is shown as SEQ ID No.4, the nucleotide sequence of the plasmid pSET152-StaR-ermE p is shown as SEQ ID No.5, and the nucleotide sequence of the plasmid pSET152-StaR-kasO p is shown as SEQ ID No. 6.
7. The method for preparing genetically engineered bacteria of claim 6, wherein the backbone of the recombinant plasmid is a pSET152 plasmid; preferably, the intermediate host bacterium is escherichia coli ET12567; preferably, the electrotransformation is performed in ISP2 agar medium at 28 ℃.
8. A process for the preparation of staurosporine, comprising fermenting the genetically engineered bacterium of any one of claims 1-4 to obtain staurosporine from the fermentation broth.
CN202310505030.7A 2023-05-06 2023-05-06 Genetically engineered bacterium for producing staurosporine and preparation method thereof Pending CN116716233A (en)

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