Dynamic regulation and control system, pantoic acid production strain as well as preparation method and application of pantoic acid production strain

Abstract

The invention discloses a dynamic regulation and control system, a pantoic acid production strain and a preparation method and application of the pantoic acid production strain. The dynamic regulation and control system comprises a LuxI module, a LuxR module, a tetR module and a tetracycline regulation and control module, wherein 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 and control module comprises a promoter Pteto for down-regulating gltA expression. Experiments prove that the dynamic regulation and control system has an obvious positive effect in the aspect of controlling the biomass accumulation of the pantoic acid producing microorganisms, not only is the problem of overhigh biomass accumulation of pantoic acid engineering bacteria in industrial production solved, but also the yield of pantoic acid is increased, and a foundation is laid for realizing industrial microbial fermentation production of pantoic acid. The method has an important application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a dynamic regulation system, a pantoic acid producing strain, and a preparation method and application thereof. Background Art

[0002] Pantothenic acid, also known as vitamin B5, is a component of coenzyme A in humans and animals and is involved in the metabolism of carbohydrates, fats and proteins. The biological synthesis pathway of pantothenic acid is the dehydration condensation of one molecule of R-pantothenic acid and one molecule of β-alanine. As an important model industrial microorganism, Escherichia coli has a wide range of applications in medicine, chemical industry, agriculture and other fields. The production of R-pantothenic acid by using Escherichia coli as a cell factory to construct an engineered strain to replace the heavy pollution production method of traditional chemical synthesis process is conducive to the technological transformation of biotechnology production of fine compounds.

[0003] The strategy of using synthetic biological tools for dynamic gene regulation in metabolic engineering is called dynamic metabolic engineering and 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 using Escherichia coli, including low activity of the key enzyme 3-methyl-2-oxobutyrate hydroxymethyltransferase, long and inefficient metabolic pathways, and difficulty in controlling fermentation production biomass. Among them, the problem of excessive accumulation of model bacteria biomass urgently needs to be solved. It not only brings unnecessary waste of carbon sources and reduces production efficiency, but also excessive biomass is not conducive to scale-up industrial production, because during the scale-up production process, the fermentation process is difficult to maintain a stable oxygen environment and nutrients in the fermenter. In addition, excessive biomass is 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 of the invention

[0005] In order to solve the above technical problems, the present invention first provides a pLux promoter, whose nucleotide sequence can be shown as SEQ ID NO:5.

[0006] The use of the pLux promoter as a sensing element in a quorum sensing system also falls within the scope of protection of the present invention. The quorum sensing system may be a Lux system.

[0007] The present invention also provides a dynamic regulation system. The dynamic regulation system includes a LuxI module, a LuxR module, a tetR module and a tetracycline regulation module;

[0008] The LuxI module includes a gene encoding LuxI protein and an expression element 1 expressing LuxI protein;

[0009] The LuxR module includes a gene encoding the LuxR protein and an expression element 2 expressing the LuxR protein;

[0010] The tetR module includes a gene encoding a repressor protein tetR and an expression element three for expressing the repressor protein tetR;

[0011] The tetracycline regulatory module includes expression element four that downregulates the expression of citrate synthase gltA.

[0012] The dynamic regulation system may specifically be composed of the LuxI module, the LuxR module, the tetR module and the tetracycline regulation module.

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

[0014] In the dynamic regulation system, the LuxR module may specifically consist of a gene encoding the LuxR protein and an expression element 2 expressing the LuxR protein.

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

[0016] In the dynamic regulation system, the tetracycline regulation module may specifically be composed of expression element four that downregulates the expression of citrate synthase gltA.

[0017] In the dynamic regulation system, expression element 1 and expression element 2 can be constitutive strong promoters. Expression element 3 and expression element 4 can be induction elements.

[0018] In the dynamic regulation system, expression element 1 may be a constitutive strong promoter M1-93, expression element 2 may be a constitutive strong promoter lpp, expression element 3 may be a promoter pLux, and expression element 4 may be a promoter Pteto.

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

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

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

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

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

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

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

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

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

[0028] In the above pantoic acid-producing recombinant bacteria, preferably, the nucleotide sequence of the promoter pLux may be as shown in SEQ ID NO:5.

[0029] In the above pantoate-producing recombinant bacteria, the starting strain may be an Escherichia coli in which the ilvE gene encoding branched-chain amino acid transaminase is weakened, the avtA gene encoding valine pyruvate aminotransferase 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 dehydratase is knocked out, and the panB gene encoding ketopantoate hydroxymethyltransferase and the panE gene encoding ketopantoate reductase are overexpressed. The starting strain may also include at least one of the sdaA gene encoding L-serine deaminase I, the sdaB gene encoding L-serine deaminase II, and the panF gene encoding pantothenate permease is knocked out.

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

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

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

[0033] In the above preparation method, the starting strain can be Escherichia coli subjected to the following modifications: weakening of the ilvE gene encoding branched-chain amino acid transaminase, knocking out of the avtA gene encoding valine pyruvate aminotransferase, knocking out of the leuDH gene encoding amino acid dehydrogenase, knocking out of the mlaZ gene encoding maltodextrin glucosidase, knocking out of the tdcB gene encoding threonine dehydratase, and overexpressing the panB gene encoding ketopantothenate hydroxymethyltransferase and the panE gene encoding ketopantothenate reductase.

[0034] In the above preparation method, the transformation of the starting strain may also include knocking out at least one of the sdaA gene encoding L-serine deaminase I, the sdaB gene encoding L-serine deaminase II, and the panF gene encoding pantothenate permease.

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

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

[0037] The present invention 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) Build a dynamic control system;

[0039] A2) constructing pantothenate-producing recombinant bacteria;

[0040] A3) production of pantoic acid;

[0041] A4) increasing the production of pantoic acid;

[0042] A5) reducing the biomass of the strain used to produce pantoic acid;

[0043] A6) Improving the production of pantoic acid and reducing the biomass of the strain used to produce pantoic acid.

[0044] The present invention also provides the application of any of the above-mentioned dynamic control systems in any of the following A2)-A6);

[0045] A2) constructing pantothenate-producing recombinant bacteria;

[0046] A3) production of pantoic acid;

[0047] A4) increasing the production of pantoic acid;

[0048] A5) reducing the biomass of the strain used to produce pantoic acid;

[0049] A6) Improving the production of pantoic acid and reducing the biomass of the strain used to produce pantoic acid.

[0050] The present invention also provides the use of any of the above pantoic acid-producing recombinant bacteria in any of the following A3)-A5);

[0051] A3) production of pantoic acid;

[0052] A4) increasing the production of pantoic acid;

[0053] A5) Reducing the biomass of a strain for producing pantoate.

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

[0055] A3) production of pantoic acid;

[0056] A4) increasing the production of pantoic acid;

[0057] A5) Reducing the biomass of a strain for producing pantoate.

[0058] The present invention also provides the use of the above method for producing pantoic acid in any of the following A4)-A6):

[0059] A4) increasing the production of pantoic acid;

[0060] A5) reducing the biomass of the strain used to produce pantoic acid;

[0061] A6) Improving the production of pantoic acid and reducing the biomass of the strain used to produce pantoic acid.

[0062] The Lux system consists of AHL, LuxI (AHL synthase), LuxR (AHL sensor activator) and Lux ​​promoter. LuxR forms a complex with AHL to activate the pLux promoter and express the repressor gene tetR. TetR will bind to the tetO promoter to inhibit the transcription of downstream resistance genes. Therefore, the inventors of this application combined the Lux system with the tetracycline regulatory system and mutated the pLux promoter to obtain a promoter pLux that is more suitable for industrial production. 7 (The nucleotide sequence is shown in SEQ ID NO: 5). Based on this, a tetR expression system that relies on quorum sensing was designed. Combined with the TCA cycle, it can control the redirection of conditional metabolic flux from the endogenous pathway to the targeted anabolic pathway. The system controls the pantoic acid-producing microorganism to switch from the growth mode to the target compound pantoic acid production mode at the appropriate stage, thereby controlling the biomass of the pantoic acid-producing microorganism and increasing the yield of the target product pantoic acid, solving the problem of excessive biomass accumulation encountered during the pilot and even amplification of pantoic acid-producing engineering bacteria, and laying an important foundation for the industrial production of pantoic acid.

[0063] Collection Instructions

[0064] Classification name: Escherichia coli

[0065] Biomaterials (strain): Sval049

[0066] Depository: China National Microbiological Culture Collection Administration General Microbiology Center

[0067] Abbreviation of depository institution: CGMCC

[0068] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0069] Date of deposit: March 6, 2020

[0070] CGMCC Registration Number: CGMCC No.19457 BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0073] Figure 3 is the OD of the fermentation broth of the Svp045 strain in Example 6 550nm Value and pantoic acid content. DETAILED DESCRIPTION

[0074] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0075] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0076] In the following examples, plasmid M1-93-LuxI, plasmid Puc57-pLux-tetR and plasmid Puc57-luxR were commissioned to Nanjing Qingke Biotechnology Co., Ltd. for synthesis, and the gene synthesis order number was NJ0069198. Plasmid M1-93-LuxI contains an M1-93-LuxI expression cassette composed of a strong promoter M1-93 and a regulatory gene luxI, plasmid Puc57-pLux-tetR contains a pLux-tetR expression cassette composed of a promoter pLux and a tetR gene, and plasmid Puc57-luxR contains an lpp-luxR expression cassette composed of a strong promoter lpp and a 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 the primers and their nucleotide sequences involved in the following examples are shown in Table 2.

[0082] Table 2

[0083]

[0084]

[0085]

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

[0087] Table 3

[0088]

[0089]

[0090] The inventors of the present invention have constructed a recombinant bacterium for producing pantoic acid after a large number of experiments. The synthesis pathway of pantoic acid is divided into four modules for research, and a variety of strategies are used to reduce biomass and increase the yield of pantoic acid. The starting strain is the Svp024A strain.

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

[0092] Strain Svp009 is obtained by strain transformation of Escherichia coli Sval049 CGMCC No.19457. For the specific construction method, please refer to the applicant's prior patent application: Application No.: 202211693007.7, Invention Name: A method for increasing the production of (R)-pantoic acid.

[0093] Example 1: Construction of LuxI module

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

[0095] The reaction system was 50 μL, including PrimeSTAR Max Premix (2×) 25 μL, Primer 1 (10 μM) 1 μL, Primer 2 (10 μM) 1 μL, Template 1 μL and ddH 2 O 22 μL.

[0096] PrimeSTAR Max Premix (2×) is derived from Max DNA Polymerase.

[0097] Reaction conditions: pre-denaturation at 98°C for 5 min; denaturation at 98°C for 10 s, annealing at 55°C for 15 s, extension at 72°C for 2.7 min, 35 cycles; extension at 72°C for 3 min; storage at 4°C.

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

[0099] 3. Place 80 μl of recombinant bacteria Svp024A-pKD46 competent cells on ice, add 100 ng of DNA fragment 1, place on ice for 2 minutes, and then transfer to the Bio-Rad electroporation cup. Use MicroPulser (Bio-Rad) electroporator, the electroporation parameter is 2.5 kv. After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporation cup, blow 5 times and transfer to a test tube, incubate overnight at 30°C and 75 rpm to obtain bacterial liquid. Apply 200 μL of bacterial liquid on LB solid culture medium containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol, culture overnight at 30°C, select single colonies for PCR verification, the primers used are primer 5 and primer 6, and the amplification product of the positive clone is a fragment of 3218 bp. Pick 3 positive clone single colonies and send them to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The single clone with correct sequencing is preserved and named Svp030 strain.

[0100] 4. Using plasmid M1-93-LuxI as a template, a primer pair consisting of primer 3 and primer 4 was used for PCR amplification (the reaction system and reaction conditions refer to the reaction system and reaction conditions in step 1, respectively), to obtain a 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. Positions 51 to 138 from the 5' end of SEQ ID NO: 1 are promoter M1-93, and positions 139 to 759 are regulatory gene luxI.

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

[0103] 6. Place 80 μl of recombinant bacterial Svp030-pKD46 competent cells on ice, add 100 ng of DNA fragment 2, place on ice for 2 minutes, and then transfer to a Bio-Rad electroporator. Use a MicroPulser electroporator with an electroporation voltage of 2.5 kV. After electroporation, quickly transfer 1 ml of LB liquid culture medium to the electroporator, blow and beat 5 times, then transfer to a test tube, and incubate overnight at 30°C and 75 rpm to obtain a bacterial solution. Transfer the bacterial solution to a flask (specification 250 ml) containing 50 ml of LB liquid culture medium containing 10% sucrose, and culture at 37°C for 24 hours; then streak on LB solid culture medium containing 6% sucrose, and culture overnight at 37°C to obtain a single colony.

[0104] 7. Each single colony was streaked on LB solid medium and LB solid medium containing 34μg / mL chloramphenicol, cultured at 37°C overnight, and single colonies that grew on LB solid medium and did not grow on LB solid medium containing 34μg / mL chloramphenicol were selected for PCR verification. The primers used were primer 5 and primer 6, and the amplification product of the positive clone was a 1395bp fragment. Three positive clone single colonies were selected and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The single clone with correct sequencing was named Svp031 strain.

[0105] Example 2: Construction of LuxR module

[0106] 1. Using plasmid pXZ-CS as a template, a primer pair consisting of primer 7 and primer 8 was used for PCR amplification (the reaction system and reaction conditions refer to the reaction system and reaction conditions in step 1 of Example 1, respectively), and a DNA fragment 3 with a size of 2719 bp was obtained. 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 re-screening marker for the second step of homologous recombination.

[0107] 2. Transform the pKD46 plasmid into 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, DNA fragment 3 was electroporated into the recombinant bacteria Svp031-pKD46 to obtain the Svp032 strain. Specifically, the competent cells of the recombinant bacteria Svp024A-pKD46 in step 3 of Example 1 were replaced with the competent cells of the recombinant bacteria Svp031-pKD46, and the DNA fragment 1 was replaced with the DNA fragment 3. The other steps remained unchanged to obtain a single colony. Single colonies were selected for PCR verification. The primers used were primers 11 and primers 12. The amplified product of the positive clone was a fragment of 3312bp. Three positive clone single colonies were selected and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The single clones with correct sequencing were preserved and named as Svp032 strain.

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

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

[0111] 5. The pKD46 plasmid was transformed into 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, DNA fragment 4 was electroporated into the recombinant bacteria Svp032-pKD46 to obtain a single colony. Specifically, the competent cells of the recombinant bacteria Svp030-pKD46 were replaced with the competent cells of the recombinant bacteria Svp032-pKD46, and the DNA fragment 2 was replaced with the DNA fragment 4, and the other steps remained unchanged.

[0113] 7. Each single colony was streaked on LB solid medium and LB solid medium containing 34μg / mL chloramphenicol, cultured at 37°C overnight, and single clones that grew on LB solid medium but did not grow on LB solid medium containing 34μg / mL chloramphenicol were selected for PCR verification. The primers used were primer 11 and primer 12, and the amplification product of the positive clone was a 1688bp fragment. Three positive clone single colonies were selected and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The single clone with correct sequencing was preserved and named Svp033 strain.

[0114] Example 3: Construction of tetR module

[0115] 1. Using plasmid pXZ-CS as a template, PCR amplification was performed using a primer pair consisting of primer 13 and primer 14 (the reaction system and reaction conditions were respectively referred to the reaction system and reaction conditions in step 1 of Example 1), and a DNA fragment 5 of 2719 bp was obtained. 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 re-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 the recombinant bacteria Svp033-pKD46.

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

[0118] 4. Using plasmid Puc57-pLux-tetR as a template, a primer pair consisting of primer 15 and primer 16 was used for PCR amplification (the reaction system and reaction conditions refer to the reaction system and reaction conditions in step 1 of Example 1, respectively), to obtain a DNA fragment 6 of 779 bp in size. The nucleotide sequence of DNA fragment 6 is shown in SEQ ID NO: 3.

[0119] DNA fragment 6 contains promoter pLux and tetR gene, wherein positions 51 to 105 from the 5' end of SEQ ID NO: 3 are promoter pLux, and positions 106 to 729 are tetR gene.

[0120] 5. The pKD46 plasmid was transformed into competent cells of the Svp034 strain to obtain the recombinant bacteria Svp034-pKD46.

[0121] 6. According to the method of step 6 in Example 1, DNA fragment 6 was electroporated into the recombinant bacteria Svp034-pKD46 to obtain a single clone. Specifically, the competent cells of the recombinant bacteria Svp030-pKD46 were replaced with the competent cells of the recombinant bacteria Svp034-pKD46, and the DNA fragment 2 was replaced with the DNA fragment 6, and the other steps remained unchanged.

[0122] 7. Each single colony was streaked on LB solid medium and LB solid medium containing 34μg / mL chloramphenicol, cultured at 37°C overnight, and single clones that grew on LB solid medium and did not grow on LB solid medium containing 34μg / mL chloramphenicol were selected for PCR verification. The primers used were primer 17 and primer 18, and the amplification product of the positive clone was a fragment of 1812bp. Three positive clone single colonies were selected and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The single clone with correct sequencing was preserved and named as Svp035 strain.

[0123] Example 4. Construction of a tetracycline regulatory module (replacing the promoter of the gltA gene with the promoter pteto)

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

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

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

[0127] 4. Using the genomic DNA of the Svp001 strain as a template, a primer pair consisting of primers 21 and 22 was used for PCR amplification (the reaction system and reaction conditions refer to the reaction system and reaction conditions in step 1 of Example 1, respectively), to obtain a DNA fragment 8 with a size of 547 bp. Using the genomic DNA of the Svp001 strain as a template, a primer pair consisting of primers 23 and 24 was used for PCR amplification (the reaction system and reaction conditions refer to the reaction system and reaction conditions in step 1 of Example 1, respectively), to obtain a DNA fragment 9 with a size of 505 bp. DNA fragments 8 and 9 were purified and mixed and used as templates, and a primer pair consisting of primers 21 and 24 was used for PCR amplification (the reaction system and reaction conditions refer to the reaction system and reaction conditions in step 1 of Example 1, respectively), to obtain a DNA fragment 10 with a nucleotide sequence as shown in SEQ ID NO: 4.

[0128] In SEQ ID NO: 4, positions 509 to 547 from the 5' end are promoter pteto, and ptetO is regulated by the repressor protein gene tetR.

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

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

[0131] 7. Each single colony was streaked on LB solid medium and LB solid medium containing 34 μg / mL chloramphenicol, and cultured at 37°C overnight. The single clones that grew on LB solid medium but did not grow on LB solid medium containing 34 μg / mL chloramphenicol were picked for PCR verification. The primers used were primer 25 and primer 26. The amplification product of the positive clone was a fragment of 1276 bp. Three positive clone single colonies were picked and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The single clone with correct sequencing was preserved and named as Svp037 strain.

[0132] Example 5: Random mutagenesis 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 refer to the reaction system and reaction conditions in step 1 of Example 1, respectively), to obtain a DNA fragment 11 with a size of 2719 bp. DNA fragment 11 has a 50 bp homologous region upstream and downstream of the pLux-tetR gene, which is used for the first step of homologous recombination and provides a re-screening marker for the second step of homologous recombination.

[0134] 2. Transform the pKD46 plasmid into competent cells of the Svp037 strain to obtain the recombinant bacteria Svp037-pKD46.

[0135] 3. According to the method of step 3 in Example 1, DNA fragment 11 was transformed into recombinant bacteria Svp037-pKD46 to obtain Svp038 strain. Specifically, the competent cells of recombinant bacteria Svp024A-pKD46 in step 3 of Example 1 were replaced with competent cells of recombinant bacteria Svp037-pKD46, DNA fragment 1 was replaced with DNA fragment 11, and other steps remained unchanged to obtain single colonies. Single colonies were selected for PCR verification, and the primers used were primers 17 and primers 18. The amplified product of the positive clone was a fragment of 4431bp. Three positive clone single colonies were selected and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The single clones with correct sequencing were preserved and named Svp038 strain.

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

[0137] According to the above method, primer 30 is replaced by primer 31-primer 44 respectively, and the other steps remain unchanged, and the amplified fragment pLux is obtained in sequence. 2 -Amplified fragment pLux 15 .

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

[0139] Fifteen primer pairs were used to amplify the promoter pLux spacer region, while the conservative region was retained.

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

[0141] 6. According to the method of step 6 in Example 1, the amplified fragment pLux 1 Electroporate into the recombinant bacteria Svp038-pKD46 to obtain a single colony. Specifically, replace the competent cells of the recombinant bacteria Svp030-pKD46 with the competent cells of the recombinant bacteria Svp038-pKD46, and replace the DNA fragment 2 with the amplified fragment pLux 1 , the other steps remain unchanged.

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

[0143] Follow the method of steps 6 and 7 above to amplify the fragment pLux 1 Replaced with the amplified fragment pLux 2 -Amplified fragment pLux 15 , the other steps remain unchanged, and the 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 are obtained in sequence.

[0144] Example 6: Production of pantoic acid by high-density fermentation of strains Svp024A, Svp037, Svp039 and Svp053

[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) was inoculated into LB solid medium for activation, and a single clone of the strain to be tested was selected and inoculated into 3 ml LB liquid medium, and cultured overnight at 37° C. and 200 rpm to obtain a primary seed culture solution.

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

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

[0149] (3) The secondary seed culture solution was inoculated into a fermentation tank (5 L) containing 2000 ml of synthetic culture medium and fermented for 72 h. Starting from the dissolved oxygen rebound point, the fermentation solution was collected every 4 h.

[0150] The setting values ​​of various parameters during the fermentation process are: 37°C, pH 7.0, dissolved oxygen 30%, ventilation flow rate 1.5vvm, stirring speed 300-1000r / min, and dissolved oxygen, stirring speed and ventilation cascade.

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

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

[0153] 2. Fermentation broth detection

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

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

[0156] (2) Take 1 mL of fermentation broth, centrifuge at 10,000 g for 1 min, and collect the supernatant.

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

[0158] (4) Using high performance liquid chromatography (HPLC) to detect the filtrate collected in step (3) to obtain the content of pantoic acid in the fermentation broth.

[0159] The HPLC detection conditions are as follows: the chromatographic column is GL Sciences Wondasil C18 chromatographic column (4.6×250mm; 5μm); mobile phase was 0.1% phosphoric acid aqueous solution: acetonitrile = 19:1; wavelength 210nm, column temperature 35°C, flow rate 1.0mL / min, injection volume 10μL.

[0160] Within 72 hours of fermentation, some test results are shown in Figure 1 (Svp024A strain), Figure 2 (Svp037 strain) and Figure 3 (Svp045 strain).

[0161] The results showed that the biomass of the Svp024A strain could reach up to 123.2; the biomass of the Svp037 strain after the introduction of the quorum sensing element was 550nm When it reaches about 10, the quorum sensing element Lux system is triggered immediately, and the signal molecule AHL triggers the pLux promoter to express the repressor protein tetR to repress the expression of the gltA gene, so that the strain tricarboxylic acid cycle cannot circulate normally. At this time, the growth of the bacteria is only about 10, which directly affects the growth of the bacteria, making it impossible for the strain to continue to ferment in the 5L tank, and pantoic acid has not begun to accumulate. The inventors of the present application speculate that the promoter pLux and the signal molecule AHL respond too sensitively, and quorum sensing can be triggered at a very low threshold.

[0162] In order to overcome the above technical bottleneck, the inventors of the present application have rationally designed and synthesized 15 mutants of the promoter pLux after a large number of experimental analyses (i.e., promoter pLux 1 ~pLux 15 ), and then through gene editing, the 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 pLux mutant were obtained in turn, and finally these strains were fermented. The fermentation results showed that the Svp045 strain had a biomass of OD 550nmWhen the OD value of Svp045 reached 30-60, the bacterial growth slowed down until it decreased. The corresponding pyruvate entered the metabolic flow of pantoic acid and began to accumulate the product pantoic acid. Finally, the production of pantoic acid reached 26.11 g / L. At this time, the OD value of Svp045 was 550nm It is only 45. Compared with the starting strain (i.e., Svp024A strain, with a maximum biomass of 123.2), the maximum biomass of Svp045 strain is 72, which is reduced by more than 50%.

[0163] It can be seen that the quorum sensing element has a significant positive effect on controlling the biomass accumulation of pantoic acid-producing microorganisms. After reasonable modification of the promoter pLux (especially the promoter pLux 7 , whose nucleotide sequence is shown in SEQ ID NO:5) is more in line with the requirements of industrial production, which not only solves the problem of excessive biomass accumulation of pantoic acid engineering bacteria in industrial production, but also increases the yield of pantoic acid, laying the foundation for the realization of industrial microbial fermentation production of pantoic acid.

[0164] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A pLux promoter, the nucleotide sequence of which is shown in SEQ ID NO:

5.

2. Use of the pLux promoter according to 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 includes a gene encoding LuxI protein and an expression element 1 expressing LuxI protein; The LuxR module includes a gene encoding the LuxR protein and an expression element 2 expressing the LuxR protein; The tetR module includes a gene encoding a repressor protein tetR and an expression element three for expressing the repressor protein tetR; The tetracycline regulatory module includes expression element 4 for down-regulating the expression of citrate synthase gltA; in, Expression element 1 and expression element 2 are constitutive strong promoters, and expression element 3 and expression element 4 are induction elements; Preferably, the first expression element is the constitutive strong promoter M1-93, the second expression element is the constitutive strong promoter lpp; the third expression element is the promoter pLux; the fourth expression element is the promoter Pteto; More preferably, the nucleotide sequence of the promoter pLux is as shown in SEQ ID NO:

5.

4. A pantoic acid-producing recombinant bacterium, which expresses LuxI protein, LuxR protein and repressor protein tetR in the starting strain and downregulates the expression of citrate synthase gltA; wherein: The repressor protein tetR is expressed under the control of the promoter pLux; the citrate synthase gltA is downregulated by the promoter Pteto; the starting strain is a strain that produces pantoate; Preferably, the nucleotide sequence of the promoter pLux is shown in SEQ ID NO:

5.

5. The pantoic acid-producing recombinant bacterium according to claim 4, characterized in that: The starting strain is an Escherichia coli in which the ilvE gene encoding branched-chain amino acid transaminase is weakened, the avtA gene encoding valine pyruvate aminotransferase 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 dehydratase is knocked out, and the panB gene encoding ketopantothenate hydroxymethyltransferase and the panE gene encoding ketopantoate reductase are overexpressed.

6. A method for preparing a pantoic acid-producing recombinant bacterium, comprising the following steps: introducing a gene encoding LuxI protein, a gene encoding LuxR protein and a gene encoding repressor protein tetR into a starting strain, and using promoter M1-93 to regulate the gene encoding LuxI protein to increase the expression level of LuxI protein, using promoter pLux to regulate the gene encoding repressor protein tetR to increase the expression level of repressor protein tetR, using promoter lpp to regulate the gene encoding LuxR protein to increase the expression level of LuxR protein, and using promoter Pteto to regulate the gene encoding citrate synthase gltA to reduce the expression level of citrate synthase gltA; the starting strain is a strain producing pantoic acid; Preferably, the nucleotide sequence of the promoter pLux is shown in SEQ ID NO:

5.

7. The preparation method according to claim 6, characterized in that: The starting strain is an Escherichia coli that has undergone the following modifications: weakening the ilvE gene encoding branched-chain amino acid transaminase, knocking out the avtA gene encoding valine pyruvate aminotransferase, knocking out the leuDH gene encoding amino acid dehydrogenase, knocking out the mlaZ gene encoding maltodextrin glucosidase, knocking out the tdcB gene encoding threonine dehydratase, and overexpressing the panB gene encoding ketopantothenate hydroxymethyltransferase and the panE gene encoding ketopantothenate reductase.

8. The preparation method according to claim 7, characterized in that: The transformation of the starting strain also includes knocking out at least one of the sdaA gene encoding L-serine deaminase I, the sdaB gene encoding L-serine deaminase II, and the panF gene encoding pantothenate permease.

9. A method for producing pantoic acid, comprising the steps of: fermenting and culturing the pantoic acid-producing recombinant bacteria according to claim 4 or 5 or the pantoic acid-producing recombinant bacteria prepared by any one of the preparation methods according to claims 6 to 8, collecting the fermentation product, and obtaining pantoic acid therefrom.

10. Use of the pLux promoter according to claim 1 in any of the following A1)-A6); Or, application of the dynamic control system according to claim 3 in any of the following A2)-A6); Or, use of the pantothenic acid-producing recombinant bacteria according to claim 4 or 5 in any of the following A3)-A5); Or, use of a pantothenic acid-producing recombinant bacterium prepared by the preparation method according to any one of claims 6 to 8 in any of the following A3)-A5); Or, use of the method for producing pantoic acid according to claim 9 in any of the following A4)-A6): A1) Build a dynamic control system; A2) constructing pantothenate-producing recombinant bacteria; A3) production of pantoic acid; A4) increasing the production of pantoic acid; A5) reducing the biomass of the strain used to produce pantoic acid; A6) Improving the production of pantoic acid and reducing the biomass of the strain used to produce pantoic acid.

Images