A dynamic regulation system, a pantoic acid-producing strain and a preparation method and application thereof

Abstract

The application discloses a dynamic regulation system, a pantolactic acid producing strain and a preparation method and application thereof. The dynamic regulation system comprises a LuxI module, a LuxR module, a tetR module and a tetracycline regulation module. The LuxI module comprises a LuxI gene and a promoter M1-93, the LuxR module comprises a LuxR gene and a promoter lpp, the tetR module comprises a tetR gene and a promoter pLux, and the tetracycline regulation module comprises a promoter Pteto for down-regulating gltA expression. Experimental results prove that the dynamic regulation system has obvious positive effects on controlling biomass accumulation of pantolactic acid producing microorganisms, solves the problem of excessive biomass accumulation of pantolactic acid engineering bacteria in industrial production, improves the pantolactic acid yield, and lays a foundation for realizing industrial microbial fermentation production of pantolactic acid. The application has important application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and particularly relates to a dynamic regulation system, a pantoic acid production strain and a preparation method and application thereof. BACKGROUND

[0002] Pantothenic acid, also known as vitamin B5, is a component of coenzyme A in human and animal bodies, and is involved in the metabolic action of carbohydrates, fats and proteins. The biosynthetic pathway of pantothenic acid is formed by dehydration condensation of 1 molecule of R-pantoic acid and 1 molecule of beta-alanine. Escherichia coli, as an important model industrial microorganism, has wide applications in medicine, chemical industry, agriculture and the like. The use of Escherichia coli as a cell factory to construct an engineering strain to produce R-pantoic acid instead of the heavy-pollution production mode of traditional chemical synthesis is conducive to realizing the technical revolution of biotechnological production of fine chemicals.

[0003] The strategy of using synthetic biological tools for dynamic gene regulation in metabolic engineering is referred to as dynamic metabolic engineering, which is common in microbial chemical production. The Lux system is one of the most studied quorum sensing systems in Vibrio fischeri.

[0004] At present, there are still many bottleneck problems in the biosynthesis of R-pantoic acid by using Escherichia coli, including low activity of the key enzyme 3-methyl-2-oxobutanoate hydroxymethyltransferase, low efficiency of the metabolic pathway, difficulty in controlling the biomass in fermentation production and the like. Among them, the problem of excessive biomass accumulation of the model bacteria urgently needs to be solved, which not only causes unnecessary waste of carbon source and reduces production efficiency, but also is not conducive to the scale-up of industrial production, because it is difficult to maintain a stable oxygen environment and nutrients in the fermentation tank in the process of scale-up production. In addition, excessive biomass is also not conducive to downstream separation and purification, which not only increases the difficulty of separation and purification, but also causes additional consumption of reagents and energy. SUMMARY

[0005] To solve the above technical problems, the present application first provides a pLux promoter, the nucleotide sequence of which can be as shown in SEQ ID NO: 5.

[0006] The application of the pLux promoter as a sensing element in a quorum sensing system also belongs to the protection scope of the present application. The quorum sensing system can be a Lux system.

[0007] The present application further provides a dynamic regulation system. The dynamic regulation system comprises a LuxI module, a LuxR module, a tetR module and a tetracycline regulation module.

[0008] The LuxI module comprises a gene encoding a LuxI protein and an expression element I for expressing the LuxI protein.

[0009] The LuxR module comprises a gene encoding a LuxR protein and an expression element two expressing the LuxR protein.

[0010] The tetR module comprises a gene encoding a repressor protein tetR and an expression element three expressing the repressor protein tetR.

[0011] The tetracycline regulation module comprises an expression element four down-regulating the expression of citrate synthase gltA.

[0012] The dynamic regulation system can specifically consist of the LuxI module, the LuxR module, the tetR module and the tetracycline regulation module.

[0013] In the dynamic regulation system, the LuxI module specifically consists of a gene encoding a LuxI protein and an expression element one expressing the LuxI protein.

[0014] In the dynamic regulation system, the LuxR module specifically consists of a gene encoding a LuxR protein and an expression element two expressing the LuxR protein.

[0015] In the dynamic regulation system, the tetR module specifically consists of a gene encoding a repressor protein tetR and an expression element three expressing the repressor protein tetR.

[0016] In the dynamic regulation system, the tetracycline regulation module specifically consists of an expression element four down-regulating the expression of citrate synthase gltA.

[0017] In the dynamic regulation system, the expression element one and the expression element two can be a constitutive strong promoter. The expression element three and the expression element four can be an inducible element.

[0018] In the dynamic regulation system, the expression element one can be a constitutive strong promoter M1-93. The expression element two can be a constitutive strong promoter lpp. The expression element three can be a promoter pLux. The expression element four can be a promoter Pteto.

[0019] More preferably, in the dynamic regulation system, the nucleotide sequence of the promoter pLux can be as shown in SEQ ID NO: 5.

[0020] The nucleotide sequence of the LuxI protein is 950-1570 of the sequence shown in GenBank: Y00509.1. The LuxI protein can be encoded by a regulatory gene luxI. In the present application, the nucleotide sequence of the regulatory gene luxI can be as shown in SEQ ID NO: 1 from the 5' end 139-759.

[0021] The nucleotide sequence of the LuxR protein is the sequence from 18 to 827 of the sequence shown in GenBank: Y00509.1. The LuxR protein can be encoded by a LuxR gene. The nucleotide sequence of the LuxR gene in the present application can be shown as SEQ ID NO: 2 from the 5' end 236 to 1045.

[0022] The nucleotide sequence of the repressor tetR is the sequence from 44 to 667 of the sequence shown in GenBank: OP581959.1. The repressor tetR can be encoded by a tetR gene. The nucleotide sequence of the tetR gene in the present application can be shown as SEQ ID NO: 3 from the 5' end 106 to 729.

[0023] The nucleotide sequence of the promoter M1-93 can be shown as SEQ ID NO: 1 from the 5' end 51 to 138.

[0024] The nucleotide sequence of the promoter lpp can be shown as SEQ ID NO: 2 from the 5' end 51 to 235.

[0025] The nucleotide sequence of the promoter pLux can be shown as SEQ ID NO: 3 from the 5' end 51 to 105.

[0026] The nucleotide sequence of the promoter Pteto can be shown as SEQ ID NO: 4 from the 5' end 509 to 547.

[0027] The present application also provides a recombinant strain for producing pantoic acid, which can express LuxI protein, LuxR protein and repressor tetR in a starting strain and down-regulate the expression of citrate synthase gltA; wherein the expression of the repressor tetR is initiated under the control of the promoter pLux; the expression of the citrate synthase gltA is down-regulated under the control of the promoter Pteto; and the starting strain is a strain for producing pantoic acid.

[0028] In the above-mentioned recombinant strain for producing pantoic acid, preferably, the nucleotide sequence of the promoter pLux can be shown as SEQ ID NO: 5.

[0029] In the above-mentioned pantoic acid-producing recombinant bacteria, the starting strain can be an Escherichia coli in which an ilvE gene encoding a branched-chain amino acid transaminase is attenuated, an avtA gene encoding a valine pyruvate aminotransferase is knocked out, a leuDH gene encoding an amino acid dehydrogenase is knocked out, a mlaZ gene encoding a maltodextrin glucosidase is knocked out, a tdcB gene encoding a threonine dehydratase is knocked out, and a panB gene encoding a ketopantoate hydroxymethyltransferase and a panE gene encoding a ketopantoate reductase are overexpressed. The starting strain can further include at least one of a sdaA gene encoding L-serine deaminase I is knocked out, a sdaB gene encoding L-serine deaminase II is knocked out, and a panF gene encoding a pantoate permease is knocked out.

[0030] In the above-mentioned pantoic acid-producing recombinant bacteria, the starting strain can be specifically the Svp024A strain mentioned in the examples.

[0031] The present application also provides a preparation method of pantoic acid-producing recombinant bacteria, which can include the following steps: introducing a gene encoding a LuxI protein, a gene encoding a LuxR protein, and a gene encoding a repressor tetR into a starting strain, and using a promoter M1-93 to regulate the gene encoding the LuxI protein to increase the expression amount of the LuxI protein, using a promoter pLux to regulate the gene encoding the repressor tetR to increase the expression amount of the repressor tetR, using a promoter lpp to regulate the gene encoding the LuxR protein to increase the expression amount of the LuxR protein, and using a promoter Pteto to regulate a gene encoding a citrate synthase gltA to reduce the expression amount of the citrate synthase gltA; the starting strain can be a pantoic acid-producing strain.

[0032] In the above-mentioned preparation method, preferably, the nucleotide sequence of the promoter pLux can be as shown in SEQ ID NO: 5.

[0033] In the above-mentioned preparation method, the starting strain can be an Escherichia coli which is attenuated in an ilvE gene encoding a branched-chain amino acid transaminase, knocked out in an avtA gene encoding a valine pyruvate aminotransferase, knocked out in a leuDH gene encoding an amino acid dehydrogenase, knocked out in a mlaZ gene encoding a maltodextrin glucosidase, knocked out in a tdcB gene encoding a threonine dehydratase, and overexpressed in a panB gene encoding a ketopantoate hydroxymethyltransferase and a panE gene encoding a ketopantoate reductase.

[0034] In the above-mentioned preparation method, the modification of the starting strain can further include at least one of knocking out a sdaA gene encoding L-serine deaminase I, knocking out a sdaB gene encoding L-serine deaminase II, and knocking out a panF gene encoding a pantoate permease.

[0035] In the above preparation method, the starting strain can be specifically the Svp024A strain mentioned in the examples.

[0036] The present application also provides a method for producing panthothenic acid, which can comprise the following steps: fermenting and culturing any of the above-mentioned panthothenic acid-producing recombinant bacteria or the panthothenic acid-producing recombinant bacteria prepared by any of the above-mentioned preparation methods, collecting the fermentation product, and obtaining panthothenic acid therefrom.

[0037] The present application also provides the use of the pLux promoter with the nucleotide sequence as shown in SEQ ID NO: 5 in any of the following A1)-A6):

[0038] A1) constructing a dynamic regulation system;

[0039] A2) constructing a panthothenic acid-producing recombinant bacterium;

[0040] A3) producing panthothenic acid;

[0041] A4) improving the yield of panthothenic acid production;

[0042] A5) reducing the biomass of the strain used for producing panthothenic acid;

[0043] A6) improving the yield of panthothenic acid production and reducing the biomass of the strain used for producing panthothenic acid.

[0044] The present application also provides the use of any of the above-mentioned dynamic regulation systems in any of the following A2)-A6):

[0045] A2) constructing a panthothenic acid-producing recombinant bacterium;

[0046] A3) producing panthothenic acid;

[0047] A4) improving the yield of panthothenic acid production;

[0048] A5) reducing the biomass of the strain used for producing panthothenic acid;

[0049] A6) improving the yield of panthothenic acid production and reducing the biomass of the strain used for producing panthothenic acid.

[0050] The present application also provides the use of any of the above-mentioned panthothenic acid-producing recombinant bacteria in any of the following A3)-A5):

[0051] A3) producing panthothenic acid;

[0052] A4) improving the yield of panthothenic acid production;

[0053] A5) reducing the biomass of the strain used for producing panthothenic acid.

[0054] The present application also provides the use of the panthothenic acid-producing recombinant bacteria prepared by any of the above-mentioned preparation methods in any of the following A3)-A5):

[0055] A3) producing panthothenate;

[0056] A4) increasing the yield of panthothenate production;

[0057] A5) reducing the biomass of the strain for producing panthothenate.

[0058] The present application also provides the use of the above-mentioned method for producing panthothenate in any of A4) to A6):

[0059] A4) increasing the yield of panthothenate production;

[0060] A5) reducing the biomass of the strain for producing panthothenate;

[0061] A6) increasing the yield of panthothenate production and reducing the biomass of the strain for producing panthothenate.

[0062] The Lux system is composed of AHL, LuxI (AHL synthetase), LuxR (AHL sensor activator) and Lux promoter, LuxR forms a complex with AHL, activates the pLux promoter, expresses the repressor gene tetR, which will bind to the tetO promoter, inhibiting the transcription of the downstream resistance gene. Therefore, the inventors of the present application combine the Lux system with the tetracycline regulation system and mutate the pLux promoter to obtain a more suitable promoter pLux 7 (nucleotide sequence as shown in SEQ ID NO: 5), based on which a quorum sensing-dependent tetR expression system is designed, which, in combination with the TCA cycle, can control the conditional metabolic flux redirection from the endogenous pathway to the targeted synthetic metabolic pathway. This system controls the switching of panthothenate-producing microorganisms from the growth mode to the target compound panthothenate production mode at the appropriate stage, thereby controlling the biomass of the panthothenate-producing microorganisms and increasing the yield of the target product panthothenate, solving the problem of excessive biomass accumulation in the pilot or even scale-up process of panthothenate-producing engineering bacteria, and laying an important foundation for realizing the industrial production of panthothenate.

[0063] DEPOSIT ACCOUNT

[0064] Classification name: Escherichia coli

[0065] Biological material (strain): Sval049

[0066] Preservation agency: China General Microbiological Culture Collection Center

[0067] Abbreviation of preservation agency: CGMCC

[0068] Address: No. 1, Beichen West Road, Beijing City, China

[0069] Date of preservation: March 6, 2020

[0070] CGMCC Registration Number: CGMCC No. 19457 Attached Figure Description

[0071] Figure 1 The OD of the fermentation broth of strain Svp024A in Example 6 550nm Value and content of pantothenic acid.

[0072] Figure 2 The OD of the fermentation broth of strain Svp037 in Example 6 550nm Value and content of pantothenic acid.

[0073] Figure 3 The OD of the fermentation broth of strain Svp045 in Example 6 550nm Value and content of pantothenic acid. Detailed Implementation

[0074] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0075] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0076] In the following examples, plasmids M1-93-LuxI, Puc57-pLux-tetR, and Puc57-luxR were all synthesized by Nanjing Qingke Biotechnology Co., Ltd., with gene synthesis order number NJ0069198. Plasmid M1-93-LuxI contains the M1-93-LuxI expression cassette composed of the strong promoter M1-93 and the regulatory gene luxI; plasmid Puc57-pLux-tetR contains the pLux-tetR expression cassette composed of the promoter pLux and the tetR gene; and plasmid Puc57-luxR contains the lpp-luxR expression cassette composed of the strong promoter lpp and the LuxR gene.

[0077] The strain names and genotypes involved in the following examples are shown in Table 1.

[0078] Table 1

[0079]

[0080]

[0081] The names of primers and their nucleotide sequences involved in the following examples are shown in Table 2.

[0082] Table 2

[0083]

[0084]

[0085]

[0086] The names of amplified fragments and their nucleotide sequences in the following examples are shown in Table 3.

[0087] Table 3

[0088]

[0089]

[0090] The inventors of the present application constructed a recombinant strain for producing panthothenic acid through a large number of experiments, which divided the synthesis pathway of panthothenic acid into four modules for research, and reduced biomass and improved the yield of panthothenic acid through various strategies. The starting strain is Svp024A strain.

[0091] The Svp024A strain used in the present application is obtained by genetic modification of strain Svp009. The construction method of strain Svp009 is described in the applicant's prior application patent: application number: 202310371764.0, and the invention name is: a ketopantoate reductase mutant and its application.

[0092] The strain Svp009 is obtained by strain modification of Escherichia coli Sval049 CGMCC No.19457. The specific construction method is described in the applicant's prior application patent: application number: 202211693007.7, and the invention name is: a method for improving the yield of (R)-panthothenic acid.

[0093] Example 1, construction of LuxI module

[0094] 1. Using plasmid pXZ-CS (recorded in the following literature: Tan, et al., Appl Environ Microbiol, 2013, 79: 4838-4844; containing cat-sacB gene) as a template, a primer pair consisting of primer 1 and primer 2 was used for PCR amplification to obtain a DNA fragment 1 with a size of 2719 bp. DNA fragment 1 has a 50 bp homologous region upstream and downstream of sdaA gene, which is used for the first step of homologous recombination to provide a counter-selection marker for the second step of homologous recombination.

[0095] The reaction system was 50 μL, including PrimeSTAR Max Premix (2x) 25 μL, primer 1 (10 μM) 1 μL, primer 2 (10 μM) 1 μL, template 1 μL and ddH2O 22 μL.

[0096] PrimeSTAR Max Premix (2x) was derived from Max DNA Polymerase.

[0097] The reaction conditions were as follows: 98 ℃ pre-denaturation for 5 min; 98 ℃ denaturation for 10 s, 55 ℃ annealing for 15 s, 72 ℃ extension for 2.7 min, 35 cycles; 72 ℃ extension for 3 min; 4 ℃ preservation.

[0098] 2. The pKD46 plasmid (Datsenko and Wanner 2000, Proc Natl Acad Sci USA 97: 6640-6645; the pKD46 plasmid was purchased from the CGSC E. coli Stock Center at Yale University, CGSC #7739) was transformed into the competent cells of Svp024A strain to obtain recombinant bacteria Svp024A-pKD46.

[0099] 3. 80 μl of recombinant bacteria Svp024A-pKD46 competent cells were placed on ice, 100 ng of DNA fragment 1 was added, and the ice was placed for 2 min, then transferred to a Bio-Rad electroporation cup. Using a MicroPulser (Bio-Rad Company) electroporation instrument, the voltage was 2.5 kv. After electroporation, 1 ml of LB liquid medium was quickly transferred to the electroporation cup, blown 5 times, then transferred to a test tube, incubated at 30 ℃, 75 rpm overnight, to obtain a bacterial solution. 200 μL of bacterial solution was smeared on LB solid medium containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol, and incubated at 30 ℃ overnight. Single colonies were selected for PCR verification, and the primers used were primer 5 and primer 6. The amplification product of the positive clone was a 3218 bp fragment. Three positive single colonies were selected for sequencing by Nanjing Qikang Biotechnology Co., Ltd. The single clone with correct sequencing was preserved and named as Svp030 strain.

[0100] 4. Using plasmid M1-93-LuxI as a template, PCR amplification was performed using primer pair consisting of primers 3 and 4 (the reaction system and reaction conditions are the same as those in step 1), resulting in DNA fragment 2 with a size of 809 bp. The nucleotide sequence of DNA fragment 2 is shown in SEQ ID NO: 1.

[0101] DNA fragment 2 contains promoter M1-93 and regulatory gene luxI. SEQ ID NO:1 contains promoter M1-93 from position 51 to 138 from the 5' end, and regulatory gene luxI from position 139 to 759.

[0102] 5. The pKD46 plasmid was transformed into competent cells of the Svp030 strain to obtain recombinant bacteria Svp030-pKD46.

[0103] 6. Place 80 μl of recombinant Svp030-pKD46 competent cells on ice, add 100 ng of DNA fragment 2, incubate on ice for 2 min, then transfer to a Bio-Rad electroporation cuvette. Use a MicroPulser electroporator with an electroporation setting of 2.5 kV. Immediately after electroporation, transfer 1 ml of LB liquid medium to the cuvette, pipette five times, then transfer to a test tube and incubate overnight at 30°C and 75 rpm to obtain the bacterial culture. Transfer the bacterial culture to a 250 ml flask containing 50 ml of LB liquid medium containing 10% sucrose and incubate at 37°C for 24 h; then streak on LB solid medium containing 6% sucrose and incubate overnight at 37°C to obtain single colonies.

[0104] 7. Streak each single colony on LB agar and LB agar containing 34 μg / mL chloramphenicol, respectively, and incubate overnight at 37°C. Select single colonies that grow on LB agar but not on LB agar containing 34 μg / mL chloramphenicol for PCR verification. Primers 5 and 6 were used. The amplification product of positive clones was a 1395 bp fragment. Three positive clones were sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The correctly sequenced single clones were preserved and named Svp031.

[0105] Example 2: Building the LuxR Module

[0106] 1. Using plasmid pXZ-CS as a template, PCR amplification was performed using primer pair consisting of primers 7 and 8 (the reaction system and conditions are as described in step 1 of Example 1), yielding DNA fragment 3 with a size of 2719 bp. DNA fragment 3 has a 50 bp homologous region upstream and downstream of the sdaB gene, which is used for the first step of homologous recombination and provides a screening marker for the second step of homologous recombination.

[0107] 2. Transform the pKD46 plasmid into the competent cells of the Svp031 strain to obtain the recombinant bacteria Svp031-pKD46.

[0108] 3. According to the method of step 3 in Example 1, electrically transform the DNA fragment 3 into the recombinant bacteria Svp031-pKD46 to obtain the Svp032 strain. Specifically, replace the recombinant bacteria Svp024A-pKD46 competent cells of step 3 in Example 1 with the recombinant bacteria Svp031-pKD46 competent cells, replace the DNA fragment 1 with the DNA fragment 3, and the other steps remain unchanged to obtain single colonies. Pick single colonies for PCR verification, and the primers used are primer 11 and primer 12. The amplification product of the positive clone is a 3312 bp fragment. Pick 3 positive clone single colonies for sequencing by Nanjing Qikeng Biotechnology Co., Ltd. The single colony with correct sequencing is preserved and named as the Svp032 strain.

[0109] 4. Use the plasmid Puc57-luxR as the template, and use the primer pair composed of primer 9 and primer 10 to perform PCR amplification (the reaction system and the reaction conditions are referred to the reaction system and the reaction conditions in step 1 in Example 1, respectively), to obtain the DNA fragment 4 with a size of 1095 bp, which is used for the second homologous recombination. The nucleotide sequence of the DNA fragment 4 is shown in SEQ ID NO: 2.

[0110] The DNA fragment 4 contains the strong promoter lpp and the LuxR gene, which is used to bind with the AHL signal molecule synthesized by the LuxI protein to activate the expression of the pLux promoter. In SEQ ID NO: 2, the positions 51-235 from the 5' end are the promoter lpp, and the positions 236-1045 are the LuxR gene.

[0111] 5. Transform the pKD46 plasmid into the competent cells of the Svp032 strain to obtain the recombinant bacteria Svp032-pKD46.

[0112] 6. According to the method of step 6 in Example 1, electrically transform the DNA fragment 4 into the recombinant bacteria Svp032-pKD46 to obtain single colonies. Specifically, replace the recombinant bacteria Svp030-pKD46 competent cells with the recombinant bacteria Svp032-pKD46 competent cells, replace the DNA fragment 2 with the DNA fragment 4, and the other steps remain unchanged.

[0113] 7. Each single colony was streaked onto LB agar and LB agar containing 34 μg / mL chloramphenicol, respectively, and incubated overnight at 37°C. Single colonies that grew on LB agar but not on LB agar containing 34 μg / mL chloramphenicol were selected for PCR verification using primers 11 and 12. The amplification product of positive clones was a 1688 bp fragment. Three positive clones were sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The correctly sequenced single colonies were preserved and named strain Svp033.

[0114] Example 3: Building the tetR module

[0115] 1. Using plasmid pXZ-CS as a template, PCR amplification was performed using primer pair consisting of primers 13 and 14 (the reaction system and conditions are as described in step 1 of Example 1), yielding DNA fragment 5 with a size of 2719 bp. DNA fragment 5 has a 50 bp homologous region upstream and downstream of the panF gene, which is used for the first step of homologous recombination and provides a screening marker for the second step of homologous recombination.

[0116] 2. The pKD46 plasmid was transformed into competent cells of the Svp033 strain to obtain recombinant bacteria Svp033-pKD46.

[0117] 3. Following the method in step 3 of Example 1, DNA fragment 5 was electroporated into recombinant bacteria Svp033-pKD46 to obtain strain Svp034. Specifically, the recombinant bacteria Svp024A-pKD46 competent cells in step 3 of Example 1 were replaced with recombinant bacteria Svp033-pKD46 competent cells, and DNA fragment 1 was replaced with DNA fragment 5. All other steps remained unchanged, resulting in single colonies. Single colonies were selected for PCR verification using primers 17 and 18. The amplification product of positive clones was a 3752 bp fragment. Three positive clones were selected and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The correctly sequenced single clones were preserved and named strain Svp034.

[0118] 4. Using plasmid Puc57-pLux-tetR as a template, PCR amplification was performed using primer pair consisting of primers 15 and 16 (the reaction system and reaction conditions are as described in step 1 of Example 1), resulting in a DNA fragment 6 with a size of 779 bp. The nucleotide sequence of DNA fragment 6 is shown in SEQ ID NO: 3.

[0119] DNA fragment 6 contains the promoter pLux and the tetR gene. In SEQ ID NO:3, the promoter pLux is located at positions 51-105 from the 5' end, and the tetR gene is located at positions 106-729.

[0120] 5. Transform the plasmid pKD46 into the competent cells of Svp034 strain to obtain the recombinant strain Svp034-pKD46.

[0121] 6. According to the method of step 6 in Example 1, electrotransform DNA fragment 6 into the recombinant strain Svp034-pKD46 to obtain a single colony. Specifically, replace the competent cells of the recombinant strain Svp030-pKD46 with the competent cells of the recombinant strain Svp034-pKD46, replace DNA fragment 2 with DNA fragment 6, and the other steps remain unchanged.

[0122] 7. Each single colony is streaked on LB solid medium and LB solid medium containing 34 μg / mL chloramphenicol, and incubated at 37°C overnight. The single colony which grows on the LB solid medium and does not grow on the LB solid medium containing 34 μg / mL chloramphenicol is picked for PCR verification, and the primers used are primer 17 and primer 18. The amplification product of the positive clone is a 1812 bp fragment. Three positive single colony clones are picked and sent to Nanjing Qikeng Biotechnology Co., Ltd. for sequencing. The single colony with correct sequencing is preserved and named as Svp035 strain.

[0123] Example 4, Construction of Tetracycline Regulatory Module (Replace the promoter of gltA gene with the promoter pteto)

[0124] 1. Use plasmid pXZ-CS as the template, and use primer 19 and primer 20 to perform PCR amplification (the reaction system and reaction conditions are referred to the reaction system and reaction conditions in step 1 of Example 1, respectively), to obtain DNA fragment 7 with a size of 2719 bp. DNA fragment 7 has a 50 bp homologous region upstream and downstream of the promoter of gltA gene, which is used for the first step of homologous recombination, and provides a screening marker for the second step of homologous recombination.

[0125] 2. Transform the plasmid pKD46 into the competent cells of Svp035 strain to obtain the recombinant strain Svp035-pKD46.

[0126] 3. Following the method in step 3 of Example 1, DNA fragment 7 was transformed into recombinant bacteria Svp035-pKD46 to obtain strain Svp036. Specifically, the recombinant bacteria Svp024A-pKD46 competent cells in step 3 of Example 1 were replaced with recombinant bacteria Svp035-pKD46 competent cells, and DNA fragment 1 was replaced with DNA fragment 7. All other steps remained unchanged, resulting in single colonies. Single colonies were selected for PCR verification using primers 25 and 26. The amplification product of positive clones was a 3856 bp fragment. Three positive clones were selected and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The correctly sequenced single clones were preserved and named strain Svp036.

[0127] 4. Using the genomic DNA of Svp001 strain as a template, PCR amplification was performed using primer pair consisting of primers 21 and 22 (the reaction system and conditions are as described in step 1 of Example 1), yielding a DNA fragment 8 of 547 bp. Using the genomic DNA of Svp001 strain as a template, PCR amplification was performed using primer pair consisting of primers 23 and 24 (the reaction system and conditions are as described in step 1 of Example 1), yielding a DNA fragment 9 of 505 bp. After purifying the products of DNA fragments 8 and 9, they were mixed and used as a template for PCR amplification using primer pair consisting of primers 21 and 24 (the reaction system and conditions are as described in step 1 of Example 1), yielding a DNA fragment 10 with the nucleotide sequence shown in SEQ ID NO: 4.

[0128] SEQ ID NO:4 The promoter pteto is located at positions 509-547 from the 5' end. ptetO is regulated by the repressor protein gene tetR.

[0129] 5. The pKD46 plasmid was transformed into competent cells of the Svp036 strain to obtain recombinant bacteria Svp036-pKD46.

[0130] 6. Following the method in step 6 of Example 1, DNA fragment 10 was electroporated into recombinant bacteria Svp036-pKD46 to obtain a single colony. Specifically, the recombinant bacteria Svp030-pKD46 competent cells were replaced with recombinant bacteria Svp036-pKD46 competent cells, DNA fragment 2 was replaced with DNA fragment 10, and all other steps remained unchanged.

[0131] 7. Each single colony was streaked on LB solid medium and LB solid medium containing 34 μg / mL chloramphenicol, respectively, and incubated at 37°C overnight. Single colonies which grew on LB solid medium but did not grow on LB solid medium containing 34 μg / mL chloramphenicol were picked and verified by PCR using primer 25 and primer 26. The positive clones were amplified to a 1276 bp fragment. Three positive single colonies were sent to Nanjing Qikeng Biotechnology Co., Ltd. for sequencing. The single colony with correct sequencing was preserved and named as Svp037 strain.

[0132] Example 5. Random mutation based on promoter pLux

[0133] 1. Using plasmid pXZ-CS as a template, a primer pair consisting of primer 27 and primer 28 was used for PCR amplification (the reaction system and reaction conditions were referred to those in step 1 of Example 1). DNA fragment 11 with a size of 2719 bp was obtained. DNA fragment 11 had a 50 bp homologous region with the upstream and downstream of pLux-tetR gene, which was used for the first step of homologous recombination and provided a secondary screening marker for the second step of homologous recombination.

[0134] 2. The pKD46 plasmid was transformed into the competent cells of Svp037 strain to obtain recombinant strain Svp037-pKD46.

[0135] 3. DNA fragment 11 was transformed into the recombinant strain Svp037-pKD46 according to the method in step 3 of Example 1 to obtain Svp038 strain. Specifically, the recombinant strain Svp024A-pKD46 competent cells in step 3 of Example 1 were replaced by the recombinant strain Svp037-pKD46 competent cells, and DNA fragment 1 was replaced by DNA fragment 11. The other steps were unchanged, and single colonies were obtained. The single colonies were selected for PCR verification using primer 17 and primer 18. The positive clones were amplified to a 4431 bp fragment. Three positive single colonies were sent to Nanjing Qikeng Biotechnology Co., Ltd. for sequencing. The single colony with correct sequencing was preserved and named as Svp038 strain.

[0136] 4. Using the genome of Svp035 strain as a template, a primer pair consisting of primer 29 and primer 30 was used for PCR amplification to obtain amplification fragment pLux 1 .

[0137] According to the above method, primer 30 was replaced by primers 31-44, respectively, and the other steps were unchanged. Amplification fragments pLux 2 , amplification fragment pLux 15 were obtained in turn.

[0138] Amplification fragment pLux 1-Amplified fragment pLux 15 The nucleotide sequences are shown in Table 3.

[0139] Fifteen primer pairs were used to mutate and amplify the pLux spacer region of the promoter, while the conserved region was preserved.

[0140] 5. The pKD46 plasmid was transformed into competent cells of the Svp038 strain to obtain recombinant bacteria Svp038-pKD46.

[0141] 6. Following the method in step 6 of Example 1, amplify the pLux fragment. 1 Electroporation was performed on recombinant bacteria Svp038-pKD46 to obtain single colonies. Specifically, Svp030-pKD46 competent cells were replaced with Svp038-pKD46 competent cells, and DNA fragment 2 was replaced with the amplified fragment pLux. 1 All other steps remain unchanged.

[0142] 7. Each single colony was streaked onto LB solid medium and LB solid medium containing 34 μg / mL chloramphenicol, respectively, and incubated overnight at 37°C. Single colonies that grew on LB solid medium but did not grow on LB solid medium containing 34 μg / mL chloramphenicol were selected for PCR verification. Primers 17 and 18 were used. The amplification product of positive clones was a 1812 bp fragment. Three positive clones were selected and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The single clones with correct sequencing were preserved and named Svp039 strain.

[0143] Following steps 6 and 7 above, the amplified fragment pLux 1 Replace them with the amplification fragment pLux respectively 2 -Amplified fragment pLux 15 With all other steps unchanged, strains Svp040, Svp041, Svp042, Svp043, Svp044, Svp045, Svp046, Svp047, Svp048, Svp049, Svp050, Svp051, Svp052, and Svp053 were obtained in sequence.

[0144] Example 6: High-density fermentation production of pantothenic acid using Svp024A, Svp037, Svp039-Svp053 strains

[0145] 1. High-density fermentation

[0146] (1) The strain to be tested (Svp024A strain, Svp037 strain, Svp039 strain, Svp040 strain, Svp041 strain, Svp042 strain, Svp043 strain, Svp044 strain, Svp045 strain, Svp046 strain, Svp047 strain, Svp048 strain, Svp049 strain, Svp050 strain, Svp051 strain, Svp052 strain or Svp053 strain) is inoculated into LB solid medium for activation, and a single colony of the strain to be tested is inoculated into 3 ml of LB liquid medium, and cultured at 37°C and 200 rpm overnight to obtain a primary seed culture.

[0147] (2) The primary seed culture is inoculated into 50 ml of synthetic medium at an inoculation amount of 5% (v / v), and cultured at 37°C and 200 rpm for 6-7 h to obtain a secondary seed culture.

[0148] The solutes and their concentrations of the synthetic medium are diammonium hydrogen phosphate 2.63 g / L, ammonium dihydrogen phosphate 0.87 g / L, potassium chloride 0.37 g / L, magnesium sulfate heptahydrate 0.37 g / L, betaine hydrochloride 0.154 g / L, AM1 trace metal salt 1.5 mL / L and glucose monohydrate 50 g / L, and the solvent is water.

[0149] (3) The secondary seed culture is inoculated into a fermenter (5 L in size) containing 2000 ml of synthetic medium, and cultured for 72 h, and the fermentation broth is taken every 4 h from the point of dissolved oxygen rebound.

[0150] The set values of various parameters during fermentation are as follows: 37°C, pH 7.0, dissolved oxygen 30%, air flow rate 1.5 vvm, stirring speed 300-1000 r / min, and dissolved oxygen, stirring speed and air flow rate are cascaded.

[0151] The feeding strategy is as follows: when the dissolved oxygen value is greater than 60%, the feeding medium is added to the fermenter, and when the dissolved oxygen value is less than 60%, the feeding is stopped.

[0152] The solutes and their concentrations of the feeding medium are 75% (m / v) glucose and 6 g / L potassium dihydrogen phosphate, and the solvent is water.

[0153] 2. Fermentation broth detection

[0154] The fermentation broth collected at different time points in step 1 is detected as follows:

[0155] (1) Take the fermentation broth and detect OD 550nm .

[0156] (2) Take 1 mL of fermentation broth, centrifuge at 10000 g for 1 min, and collect the supernatant.

[0157] (3) The supernatant collected in step (2) is diluted 20 times with sterile water, and then filtered through a 0.22 μm filter membrane to collect the filtrate.

[0158] (4) The filtrate collected in step (3) is detected by high performance liquid chromatography (HPLC) to obtain the content of protocatechuate in the fermentation broth.

[0159] The detection conditions of high performance liquid chromatography (HPLC) are as follows: the chromatographic column is GL Sciences Wondasil C18 chromatographic column (4.6x250mm; 5μm); mobile phase is 0.1% phosphoric acid aqueous solution: acetonitrile = 19:1; wavelength 210nm, column temperature 35℃, flow rate 1.0mL / min, injection volume 10μL.

[0160] Part of the detection results within 72h of fermentation are shown in Table 1. Figure 1 (Svp024A strain), Figure 2 (Svp037 strain) and Figure 3 (Svp045 strain).

[0161] The results show that the biomass of Svp024A strain can reach 123.2; the biomass of Svp037 strain after introducing the quorum sensing element is about 10, which directly affects the growth of the strain, so that the strain cannot continue to be fermented in a 5L tank, and protocatechuate does not start to accumulate. 550nm When the OD reaches about 10, the quorum sensing element Lux system is triggered immediately, the signal molecule AHL triggers the expression of the repressor protein tetR to repress the expression of the gltA gene, so that the tricarboxylic acid cycle cannot circulate normally, and the growth of the strain is only about 10, which directly affects the growth of the strain, so that the strain cannot continue to be fermented in a 5L tank, and protocatechuate does not start to accumulate. The inventors of the present application speculate that the promoter pLux and the signal molecule AHL are too sensitive in response, and can trigger quorum sensing at a very low threshold.

[0162] To break through the above technical bottleneck, the inventors of the present application have carried out a large number of experimental analyses, reasonably designed and synthesized 15 mutants of the promoter pLux (i.e. promoters pLux 1 ~ pLux 15 ), and then obtained Svp039 strain, Svp040 strain, Svp041 strain, Svp042 strain, Svp043 strain, Svp044 strain, Svp045 strain, Svp046 strain, Svp047 strain, Svp048 strain, Svp049 strain, Svp050 strain, Svp051 strain, Svp052 strain and Svp053 strain expressing the above pLux mutants by gene editing, and finally fermented these strains. The fermentation results show that the biomass of Svp045 strain is about 123.2, which is the highest among the strains.550nm responded between 30 to 60, the growth of the bacteria slowed down until it decreased, and the corresponding pyruvic acid entered the metabolic flow of the pantoic acid to start accumulating the product pantoic acid, and finally the yield of pantoic acid reached 26.11 g / L, at this time the OD 550nm was only 45, and compared with the starting strain (i.e. Svp024A strain, the highest biomass was 123.2), the highest biomass of Svp045 strain was 72, which was reduced by more than 50%.

[0163] It can be seen that the quorum sensing element has obvious positive effect on controlling the biomass accumulation of pantoic acid-producing microorganisms, and after reasonable modification of the promoter pLux (especially the promoter pLux 7 with the nucleotide sequence as shown in SEQ ID NO: 5), it is more suitable for industrial production, not only solves the problem of high biomass accumulation of pantoic acid engineering bacteria in industrial production, but also improves the yield of pantoic acid, and lays a foundation for realizing the industrial microbial fermentation production of pantoic acid.

[0164] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In short, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the scope disclosed in the present application.

Claims

1. A pLux promoter, whose nucleotide sequence is shown in SEQ ID NO:

5. 2.Use of the pLux promoter of claim 1 as a sensing element in a quorum sensing system. 3.A dynamic regulation system comprising a LuxI module, a LuxR module, a tetR module and a tetracycline regulation module; The LuxI module comprises a gene encoding a LuxI protein and an expression element one for expressing the LuxI protein; The LuxR module comprises a gene encoding a LuxR protein and an expression element two for expressing the LuxR protein; The tetR module comprises a gene encoding a repressor protein tetR and an expression element three for expressing the repressor protein tetR; The tetracycline regulation module comprises an expression element four for down-regulating the expression of citrate synthase gltA; wherein The expression element one and the expression element two are constitutive strong promoters, and the expression element three and the expression element four are sensing elements; The expression element one is a constitutive strong promoter M1-93, the expression element two is a constitutive strong promoter lpp, the expression element three is a promoter pLux, and the expression element four is a promoter Pteto; The nucleotide sequence of the promoter pLux is shown in SEQ ID NO:

5.

4. A recombinant organism producing pantoic acid, which is a starting strain expressing Luxl protein, LuxR protein and repressor protein tetR and down-regulating expression of citrate synthase gltA; wherein, The repressor protein tetR is under the control of the promoter pLux to initiate expression, and the citrate synthase gltA is under the control of the promoter Pteto to be down-regulated, and the starting strain is a strain for producing pantoic acid; The nucleotide sequence of the promoter pLux is shown in SEQ ID NO:

5. The starting strain is an Escherichia coli strain in which an ilvE gene encoding a branched-chain amino acid transaminase is attenuated, an avtA gene encoding a valine pyruvate amino transferase is knocked out, a leuDH gene encoding an amino acid dehydrogenase is knocked out, a mlaZ gene encoding a maltodextrin glucosidase is knocked out, a tdcB gene encoding a threonine dehydrase is knocked out, and a panB gene encoding a ketopantoate hydroxymethyltransferase and a panE gene encoding a ketopantoic acid reductase are overexpressed; The modification of the starting strain further comprises at least one of knocking out a sdaA gene encoding an L-serine deaminase I, knocking out a sdaB gene encoding an L-serine deaminase II, and knocking out a panF gene encoding a pantoate permease. 5.A method for preparing a pantoic acid-producing recombinant strain, comprising the following steps: introducing a gene encoding a LuxI protein, a gene encoding a LuxR protein and a gene encoding a repressor protein tetR into a starting strain, and using a promoter M1-93 to regulate the gene encoding the LuxI protein to increase the expression amount of the LuxI protein, using a promoter pLux to regulate the gene encoding the repressor protein tetR to increase the expression amount of the repressor protein tetR, using a promoter lpp to regulate the gene encoding the LuxR protein to increase the expression amount of the LuxR protein, and using a promoter Pteto to regulate the gene encoding a citrate synthase gltA to decrease the expression amount of the citrate synthase gltA; the starting strain is a strain for producing pantoic acid; The nucleotide sequence of the promoter pLux is shown in SEQ ID NO:

5. The starting strain is an Escherichia coli which is modified as follows: the ilvE gene encoding branched-chain amino acid transaminase is attenuated, the avtA gene encoding valine pyruvate amino transferase is knocked out, the leuDH gene encoding amino acid dehydrogenase is knocked out, the mlaZ gene encoding maltodextrin glucosidase is knocked out, the tdcB gene encoding threonine dehydrase is knocked out, and the panB gene encoding ketopantoate hydroxymethyltransferase and the panE gene encoding ketopantoate reductase are overexpressed; The modification of the starting strain further comprises at least one of the following: the sdaA gene encoding L-serine deaminase I is knocked out, the sdaB gene encoding L-serine deaminase II is knocked out, and the panF gene encoding pantoate permease is knocked out.

6. A method for producing pantoic acid, comprising the following steps: fermenting and culturing the pantoic acid-producing recombinant bacteria of claim 4 or the pantoic acid-producing recombinant bacteria prepared by the preparation method of claim 5, collecting the fermentation product, and obtaining pantoic acid therefrom.

7. Use of the pLux promoter of claim 1 in any of the following A1) to A6): A1) constructing a dynamic regulation system; A2) constructing pantoic acid-producing recombinant bacteria; A3) producing pantoic acid; A4) increasing the yield of pantoic acid production; A5) reducing the biomass of the strain used for producing pantoic acid; A6) increasing the yield of pantoic acid production and reducing the biomass of the strain used for producing pantoic acid.

8. Use of the dynamic regulation system of claim 3 in any of the following A2) to A6): A2) constructing pantoic acid-producing recombinant bacteria; A3) producing pantoic acid; A4) increasing the yield of pantoic acid production; A5) reducing the biomass of the strain used for producing pantoic acid; A6) increasing the yield of pantoic acid production and reducing the biomass of the strain used for producing pantoic acid.

9. Use of the pantoic acid-producing recombinant bacteria of claim 4 in any of the following A3) to A5): A3) producing pantoic acid; A4) increasing the yield of pantoic acid production; A5) reducing the biomass of the strain used for producing pantoic acid.

10. Use of the pantoic acid-producing recombinant bacteria prepared by the preparation method of claim 5 in any of the following A3) to A5): A3) producing pantoic acid; A4) increasing the yield of pantoic acid production; A5) reducing the biomass of the strain used for producing pantoic acid.

11. Use of the method for producing pantoic acid of claim 6 in any of the following A4) to A6): A4) increasing the yield of pantoic acid production; A5) reducing the biomass of the strain used for producing pantoic acid; A6) increasing the yield of pantoic acid production and reducing the biomass of the strain used for producing pantoic acid.

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