CN119709733A - Dynamic regulation system, pantoic acid production strain, preparation method and application thereof - Google Patents

Abstract

The invention discloses a dynamic regulation system, a pantoic acid production strain, 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, 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 module comprises a promoter Pteto for down-regulating gltA expression. Experiments prove that the dynamic regulation and control system has obvious positive effects in the aspect of controlling biomass accumulation of microorganisms producing pantoic acid, not only solves the problem of excessively 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 fermentation production of pantoic acid by industrial microorganisms. The invention has important application value.

Description

Dynamic regulation system, pantoic acid production strain, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a dynamic regulation system, a pantoic acid production strain, a preparation method and application thereof.

Background

Pantothenic acid (Pantothenic acid), also known as vitamin B5, is a constituent of coenzyme a in humans and animals and is involved in carbohydrate, fat and protein metabolism. The biosynthesizing route of pantothenic acid is formed by dehydrating and condensing 1 molecule of R-pantoic acid and 1 molecule of beta-alanine. Coli is an important model industrial microorganism and has wide application in medicine, chemical industry, agriculture and the like. The engineering strain constructed by using the escherichia coli as a cell factory is used for producing R-pantoic acid to replace a heavy pollution production mode of the traditional chemical synthesis process, so that the technical transformation of producing the fine compound by using a biotechnology is facilitated.

Strategies for dynamic gene regulation using synthetic biological tools in metabolic engineering are called dynamic metabolic engineering and are common in biochemical production of microorganisms. The Lux system is one of the quorum sensing systems currently being studied in vibrio freudenreichii.

At present, the biosynthesis of R-pantoic acid by using escherichia coli has a plurality of bottleneck problems including low activity of key enzyme 3-methyl-2-oxo-butyrate hydroxymethyl transferase, low long-term efficiency of metabolic pathway, difficult control of fermentation production biomass and the like. The problem that the biomass accumulation of the model bacteria is too high is urgent to be solved, so that not only is unnecessary carbon source waste brought about and the production efficiency is reduced, but also the excessive biomass is unfavorable for the scale-up industrial production, because the fermentation process is difficult to maintain stable oxygen environment and nutrient substances in the fermentation tank in the scale-up production process. In addition, the excessive biomass is not favorable for downstream separation and purification, not only improves the difficulty of separation and purification, but also causes additional consumption of reagents and energy.

Disclosure of Invention

In order to solve the technical problems, the invention firstly provides pLux promoter, and the nucleotide sequence of the pLux promoter can be shown as SEQ ID NO. 5.

The use of the pLux promoter as a sensing element in quorum sensing systems is also within the scope of the present invention. The quorum sensing system may be a Lux system.

The invention also provides a dynamic regulation and control system. The dynamic regulation and control system comprises a LuxI module, a LuxR module, a tetR module and a tetracycline regulation and control module;

the LuxI module comprises a gene for encoding the LuxI protein and an expression element I for expressing the LuxI protein;

The LuxR module comprises a gene for encoding the LuxR protein and an expression element II for expressing the LuxR protein;

The tetR module comprises a gene encoding a repressor tetR and an expression element III for expressing the repressor tetR;

the tetracycline regulatory module comprises an expression element IV for down-regulating the expression of the citrate synthase gltA.

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

In the dynamic regulation system, the LuxI module may specifically consist of a gene encoding LuxI protein and an expression element expressing LuxI protein.

In the dynamic regulation system, the LuxR module can specifically consist of a gene for encoding the LuxR protein and an expression element II for expressing the LuxR protein.

In the dynamic regulation system, the tetR module may specifically consist of a gene encoding the repressor tetR and an expression element three for expressing the repressor tetR.

In the dynamic regulation system, the tetracycline regulation module can specifically consist of an expression element IV for down-regulating the expression of the citrate synthase gltA.

In the dynamic regulation system, the first expression element and the second expression element can be constitutive strong promoters. Expression element three and expression element four may be sensing elements.

In the dynamic regulation system, the first expression element can be a constitutive strong promoter M1-93. The expression element two may be a constitutive strong promoter lpp. Expression element three may be a promoter pLux. Expression element four may be a promoter Pteto.

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

The nucleotide sequence of the LuxI protein is 950-1570 of a sequence shown in GenBank:Y 00509.1. The LuxI protein may be encoded by the regulatory gene LuxI. The nucleotide sequence of the regulatory gene luxI can be shown as 139-759 positions from the 5' -end of SEQ ID NO. 1.

The nucleotide sequence of the LuxR protein is 18-8237 th sequence of a sequence shown in GenBank: Y00509.1. The LuxR protein may be encoded by the LuxR gene. The nucleotide sequence of the LuxR gene in the invention can be shown as 236-1045 th position from the 5' end of SEQ ID NO. 2.

The nucleotide sequence of the repressor tetR is the 44 th-667 th sequence of the sequence shown in GenBank: OP 581959.1. The repressor tetR may be encoded by the tetR gene. The nucleotide sequence of the tetR gene in the invention can be shown as 106-729 th position from the 5' end of SEQ ID NO. 3.

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

The nucleotide sequence of the promoter lpp can be shown as 51-235 positions from the 5' end of SEQ ID NO. 2.

The nucleotide sequence of promoter pLux can be shown as SEQ ID NO. 3 from position 51-105 of the 5' end.

The nucleotide sequence of promoter Pteto can be shown as SEQ ID NO. 4 from position 509-547 at the 5' end.

The invention also provides a recombinant bacterium for producing pantoic acid, which can express LuxI protein, luxR protein and repressor protein tetR in a starting strain body and down-regulate the expression of the citrate synthase gltA, wherein the repressor protein tetR is used for promoting the expression under the control of a promoter pLux, 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.

In the recombinant bacterium for producing pantoic acid, preferably, the nucleotide sequence of the promoter pLux can be shown as SEQ ID NO. 5.

In the recombinant bacterium for producing pantoic acid, the starting strain may be escherichia coli which weakens ilvE gene encoding branched-chain amino acid transaminase, knocks out avtA gene encoding valine pyruvic acid aminotransferase, knocks out leuDH gene encoding amino acid dehydrogenase, knocks out mlaZ gene encoding maltodextrin glucosidase, knocks out tdcB gene encoding threonine dehydratase and overexpresses panB gene encoding ketopantothenate hydroxymethyltransferase and panE gene encoding ketopantoic acid reductase. The starting strain may further comprise at least one of a knock-out of the sdaA gene encoding L-serine deaminase I, a knock-out of the sdaB gene encoding L-serine deaminase II, and a knock-out of the panF gene encoding pantothenate permease.

In the recombinant strain for producing pantoic acid, the starting strain can be specifically an Svp024A strain mentioned in the example.

The invention also provides a preparation method of the pantoic acid-producing recombinant bacterium, which comprises the steps of introducing a gene encoding LuxI protein, a gene encoding LuxR protein and a gene encoding repressor protein tetR into a starting strain, regulating and controlling the gene encoding the LuxI protein by using promoters M1-93 to improve the expression quantity of the LuxI protein, regulating and controlling the gene encoding repressor protein tetR by using promoters pLux to improve the expression quantity of the repressor protein tetR, regulating and controlling the gene encoding the LuxR protein by using a promoter lpp to improve the expression quantity of the LuxR protein, regulating and controlling the gene encoding citrate synthase gltA by using a promoter Pteto to reduce the expression quantity of citrate synthase gltA, wherein the starting strain can be a strain for producing pantoic acid.

In the above preparation method, preferably, the nucleotide sequence of the promoter pLux is shown in SEQ ID NO. 5.

In the above preparation method, the starting strain may be E.coli modified by weakening ilvE gene encoding branched-chain amino acid transaminase, knocking out avtA gene encoding valine pyruvic aminotransferase, knocking out leuDH gene encoding amino acid dehydrogenase, knocking out mlaZ gene encoding maltodextrin glucosidase, knocking out tdcB gene encoding threonine dehydratase, and overexpressing panB gene encoding ketopantothenate hydroxymethyltransferase and panE gene encoding ketopantoate reductase.

In the above preparation method, the modification of the starting strain may further comprise at least one of knocking out the sdaA gene encoding L-serine deaminase I, knocking out the sdaB gene encoding L-serine deaminase II, and knocking out the panF gene encoding pantothenate permease.

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

The invention also provides a method for producing the pantoic acid, which can comprise the following steps of fermenting and culturing any recombinant bacterium producing the pantoic acid or the recombinant bacterium producing the pantoic acid prepared by any preparation method, and collecting a fermentation product to obtain the pantoic acid.

The invention also provides application of the pLux promoter with the nucleotide sequence shown in SEQ ID NO. 5 in any one of the following A1) to A6);

A1 A dynamic regulation system is constructed;

A2 Constructing recombinant bacteria for producing pantoic acid;

a3 Producing pantoic acid;

a4 Improving the yield of pantoic acid;

A5 Reducing the biomass of the strain used to produce pantoic acid;

a6 Increased production of pantoic acid and decreased biomass of the strain used to produce pantoic acid.

The invention also provides an application of any one of the dynamic regulation and control systems in any one of the following A2) to A6);

A2 Constructing recombinant bacteria for producing pantoic acid;

a3 Producing pantoic acid;

a4 Improving the yield of pantoic acid;

A5 Reducing the biomass of the strain used to produce pantoic acid;

a6 Increased production of pantoic acid and decreased biomass of the strain used to produce pantoic acid.

The invention also provides application of any one of the recombinant bacteria for producing pantoic acid in any one of the following A3) to A5);

a3 Producing pantoic acid;

a4 Improving the yield of pantoic acid;

A5 Reducing the biomass of the strain used to produce pantoic acid.

The invention also provides application of the pantoic acid-producing recombinant bacterium prepared by any one of the preparation methods in any one of the following A3) to A5);

a3 Producing pantoic acid;

a4 Improving the yield of pantoic acid;

A5 Reducing the biomass of the strain used to produce pantoic acid.

The invention also provides the application of the method for producing pantoic acid in any one of the following A4) to A6):

a4 Improving the yield of pantoic acid;

A5 Reducing the biomass of the strain used to produce pantoic acid;

a6 Increased production of pantoic acid and decreased biomass of the strain used to produce pantoic acid.

The Lux system consists of AHL, luxI (AHL synthase), luxR (AHL sensor activator) and Lux promoter, which form a complex with AHL, activate pLux promoter, express repressor gene tetR, which will bind to tetO promoter and repress transcription of downstream resistance gene. Therefore, the inventor of the application combines the Lux system with the tetracycline regulation system and mutates pLux promoter to obtain a promoter pLux ⁷ (the nucleotide sequence is shown as SEQ ID NO: 5) which is more suitable for industrial production, and designs a tetR expression system which depends on quorum sensing based on the promoter pLux ⁷, and can control the redirection of conditional metabolic flux from an endogenous pathway to a targeted anabolic pathway in combination with the TCA cycle. The system controls the pantoic acid production microorganism to be switched from a growth mode to a target compound pantoic acid production mode at a proper stage, thereby controlling the biomass of the pantoic acid production microorganism, improving the yield of the pantoic acid of a target product, solving the problem of excessive biomass accumulation in the pilot scale test or even amplification process of the pantoic acid production engineering bacteria and laying an important foundation for realizing industrial production of pantoic acid.

Preservation description

Classification and naming of Escherichia coli ESCHERICHIA COLI

Biological material (strain) Sval049

Preservation institution China general microbiological culture Collection center (China Committee for culture Collection of microorganisms)

The preservation organization is called CGMCC for short

Address, beijing, chaoyang district North Star, court 1, no. 3

The preservation date is 3/6 of 2020

Registration number of collection center CGMCC No.19457

Drawings

FIG. 1 shows the OD _550nm and pantoic acid content of the fermentation broth of the Svp024A strain of example 6.

FIG. 2 shows the OD _550nm and the pantoic acid content of the fermentation broth of the Svp037 strain of example 6.

FIG. 3 shows the OD _550nm values and the pantoic acid content of the fermentation broth of the Svp045 strain of example 6.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

In the examples described below, plasmid M1-93-LuxI, plasmid Puc57-pLux-tetR and plasmid Puc57-luxR were all assigned to the synthesis by Nanjing, the biological sciences Co., ltd, and the order of the gene synthesis was NJ0069198. Wherein the plasmid M1-93-luxI comprises a strong promoter M1-93 and a M1-93-luxI expression cassette composed of a regulatory gene luxI, the plasmid Puc57-pLux-tetR comprises a promoter pLux and a pLux-tetR expression cassette composed of a tetR gene, and the plasmid Puc57-luxR comprises a strong promoter lpp and a lpp-luxR expression cassette composed of a luxR gene.

The names and genotypes of the strains involved in the following examples are shown in Table 1.

TABLE 1

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

TABLE 2

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

TABLE 3 Table 3

The inventor of the invention constructs recombinant bacteria for producing pantoic acid through a large number of experiments, divides the synthesis path of pantoic acid into four modules for research, reduces biomass and improves the yield of pantoic acid through various strategies. Wherein the starting strain is Svp024A strain.

The Svp024A strain used in the invention is obtained by genetic modification of a strain Svp009, and a construction method of the strain Svp009 is shown in the prior application patent of the applicant, the application number is 202310371764.0, and the invention is named as a ketopantoate reductase mutant and application thereof.

The strain Svp009 is obtained by modifying Escherichia coli ESCHERICHIA COLI SVAL049 CGMCC No.19457 by the strain, and the specific construction method is shown in the prior application patent of the applicant, the application number is 202211693007.7, and the invention name is a method for improving the yield of (R) -pantoic acid.

Example 1 construction of LuxI Module

1. The plasmid pXZ-CS (described in the following documents: tan, et al, applEnvironMicrobiol,2013,79:4838-4844; containing the cat-sacB gene) was used as a template, and PCR amplification was performed using a primer pair consisting of primer 1 and primer 2, to obtain a DNA fragment 1 having a size of 2719 bp. The DNA fragment 1 has 50bp homologous region with the upstream and downstream of the sdaA gene, and is used for the first step of homologous recombination and provides a rescreen marker for the second step of homologous recombination.

The reaction system was 50. Mu.L, including PRIMESTAR MAX PREMIX (2X) 25. Mu.L, 1. Mu.L of primer 1 (10. Mu.M), 1. Mu.L of primer 2 (10. Mu.M), 1. Mu.L of template and 22. Mu.L of ddH ₂ O.

PRIMESTAR MAX PREMIX (2X) is derived fromMax DNAPolymerase。

The reaction conditions were 98℃pre-denaturation for 5min, 98℃denaturation for 10s,55℃annealing for 15s,72℃extension for 2.7min,35 cycles, 72℃extension for 3min, 4℃storage.

2. The pKD46 plasmid (DatsenkoandWanner, proc NatlAcadSciUSA97:6640-6645; pKD46 plasmid was purchased from the university of U.S. CGSC E.coli collection, CGSC # 7739) was transformed into competent cells of Svp024A strain to give recombinant Svp024A-pKD46.

3. Mu.l of recombinant Svp024A-pKD46 competent cells were placed on ice, 100ng of DNA fragment 1 was added, and placed on ice for 2min, and then transferred to a Bio-Rad cuvette. An electroporation apparatus (Bio-Rad Co.) was used, and the electric shock parameter was 2.5kv. 1ml of LB liquid medium is quickly transferred to a shocking cup after shocking, transferred to a test tube after 5 times of shocking, and incubated at 30 ℃ and 75rpm overnight to obtain bacterial liquid. 200 mu L of bacterial liquid is smeared on LB solid medium containing 100 mu g/mL of ampicillin and 34 mu g/mL of chloramphenicol, cultured overnight at 30 ℃, single colony is selected for PCR verification, the primers are primer 5 and primer 6, and the amplified product of positive clone is a fragment of 3218 bp. 3 positive clone single colonies were picked and sent to Nanjing qing department biotechnology Co., ltd for sequencing, and the correctly sequenced single clone was preserved and named Svp030 strain.

4. The plasmid M1-93-LuxI is used as a template, and a primer pair consisting of a primer 3 and a primer 4 is adopted for PCR amplification (a reaction system and a reaction condition are respectively referred to the reaction system and the reaction condition in the step 1), so that the DNA fragment 2 with 809bp size is obtained. The nucleotide sequence of the DNA fragment 2 is shown as SEQ ID NO. 1.

The DNA fragment 2 contains the promoter M1-93 and the regulatory gene luxI, and the 51 st to 138 th positions from the 5' end of the SEQ ID NO. 1 are the promoters M1-93 and the 139 th to 759 th positions are the regulatory genes luxI.

5. The pKD46 plasmid was transformed into competent cells of Svp030 strain to obtain recombinant Svp030-pKD46.

6. Mu.l of recombinant Svp030-pKD46 competent cells were placed on ice, 100ng of DNA fragment 2 was added, and placed on ice for 2min before transfer to Bio-Rad cuvette. The electric shock parameters were 2.5kv using a MicroPulser electroporation apparatus. 1ml of LB liquid medium is quickly transferred to a shocking cup after shocking, transferred to a test tube after 5 times of shocking, and incubated at 30 ℃ and 75rpm overnight to obtain bacterial liquid. The bacterial liquid was transferred to a flask (250 ml in size) containing 50ml of LB liquid medium containing 10% sucrose, cultured at 37℃for 24 hours, and then streaked on LB solid medium containing 6% sucrose, cultured at 37℃overnight to give single colonies.

7. Each single colony was streaked on LB solid medium and LB solid medium containing 34. Mu.g/mL chloramphenicol, respectively, cultured overnight at 37℃and single colonies grown on LB solid medium and not grown on LB solid medium containing 34. Mu.g/mL chloramphenicol were picked for PCR verification using primers 5 and 6, and the amplified product of the positive clone was a 1395bp fragment. 3 positive clone single colonies were selected and sent to Nanjing qing department biotechnology Co., ltd for sequencing, and the correct monoclonal bacteria were sequenced and named Svp031 strain.

Example 2 construction of LuxR Module

1. The plasmid pXZ-CS was used as a template, and PCR amplification was performed using a primer set consisting of primer 7 and primer 8 (reaction system and reaction conditions were respectively referred to the reaction system and reaction conditions in step 1 of example 1), to obtain a DNA fragment 3 having a size of 2719 bp. The DNA fragment 3 has 50bp homologous region with the upstream and downstream of the sdaB gene, and is used for the first step of homologous recombination and provides a rescreen marker for the second step of homologous recombination.

2. The pKD46 plasmid is transformed into competent cells of Svp031 strain to obtain recombinant bacteria Svp031-pKD46.

3. The DNA fragment 3 was electrically transferred to recombinant Svp031-pKD46 in the same manner as in step 3 of example 1 to obtain Svp032 strain. Specifically, the competent cells of the recombinant bacteria Svp024A-pKD46 in the step 3 in the example 1 are replaced by competent cells of the recombinant bacteria Svp031-pKD46, the DNA fragment 1 is replaced by the DNA fragment 3, and other steps are unchanged, so that single colony is obtained. Single colonies were selected for PCR verification using primers 11 and 12, and the amplified product of the positive clone was a 3312bp fragment. 3 positive clone single colonies are picked and sent to Nanjing qing department biotechnology Co-Ltd for sequencing, and the monoclonal bacteria with correct sequencing is preserved and named as Svp032 strain.

4. The plasmid Puc57-luxR is used as a template, and a primer pair consisting of a primer 9 and a primer 10 is used for PCR amplification (a reaction system and a reaction condition are respectively referred to the reaction system and the reaction condition in the step 1 of the example 1), so that a DNA fragment 4 with the size of 1095bp is obtained and used for the second homologous recombination. The nucleotide sequence of the DNA fragment 4 is shown as SEQ ID NO. 2.

DNA fragment 4 contains the strong promoter lpp and the LuxR gene for binding to the AHL signal molecule synthesized by the LuxI protein to activate pLux promoter expression, wherein the 51-235 th site from the 5' end of SEQ ID NO. 2 is the promoter lpp, and the 236-1045 th site is the LuxR gene.

5. The pKD46 plasmid is transformed into competent cells of Svp032 strain to obtain recombinant bacteria Svp032-pKD46.

6. The DNA fragment 4 was electrotransferred into recombinant Svp032-pKD46 in the same manner as in step 6 of example 1, to obtain a single colony. Specifically, the competent cells of recombinant bacteria Svp030-pKD46 are replaced by competent cells of recombinant bacteria Svp032-pKD46, the DNA fragment 2 is replaced by the DNA fragment 4, and other steps are unchanged.

7. Each single colony was streaked on LB solid medium and LB solid medium containing 34. Mu.g/mL chloramphenicol, respectively, cultured overnight at 37℃and the single clone grown on LB solid medium but not on LB solid medium containing 34. Mu.g/mL chloramphenicol was picked for PCR verification using primers 11 and 12, and the amplified product of the positive clone was 1688bp fragment. 3 positive clone single colonies were picked and sent to Nanjing qing department biotechnology Co., ltd for sequencing, and the correctly sequenced single clone was preserved and named as Svp033 strain.

Example 3 construction of tetR Module

1. The plasmid pXZ-CS was used as a template, and PCR was performed using a primer set consisting of primer 13 and primer 14 (reaction system and reaction conditions were respectively referred to the reaction system and reaction conditions in step 1 of example 1), to obtain a DNA fragment 5 having a size of 2719 bp. The DNA fragment 5 has 50bp homologous region with the upstream and downstream of panF gene, which is used for the first step of homologous recombination and provides a rescreen mark for the second step of homologous recombination.

2. The pKD46 plasmid was transformed into competent cells of Svp033 strain to obtain recombinant Svp033-pKD46.

3. The DNA fragment 5 was electrotransferred to recombinant Svp033-pKD46 as in step 3 of example 1 to obtain Svp034 strain. Specifically, the competent cells of the recombinant bacteria Svp024A-pKD46 in the step 3 in the example 1 are replaced by competent cells of the recombinant bacteria Svp033-pKD46, the DNA fragment 1 is replaced by the DNA fragment 5, and other steps are unchanged, so that single colony is obtained. Single colonies were selected for PCR verification using primers 17 and 18, and the amplified product of the positive clone was a 3752bp fragment. 3 positive clone single colonies were picked and sent to Nanjing qing department biotechnology Co., ltd for sequencing, and the correctly sequenced single clone was preserved and named as Svp034 strain.

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

DNA fragment 6 contains promoter pLux and tetR gene, wherein SEQ ID NO. 3 shows promoter pLux at positions 51-105 from the 5' end and tetR gene at positions 106-729.

5. The pKD46 plasmid was transformed into competent cells of Svp034 strain to obtain recombinant Svp034-pKD46.

6. The DNA fragment 6 was electrically transferred into recombinant Svp034-pKD46 in the same manner as in step 6 of example 1 to obtain a monoclonal antibody. Specifically, the competent cells of the recombinant bacteria Svp030-pKD46 are replaced by competent cells of the recombinant bacteria Svp034-pKD46, the DNA fragment 2 is replaced by the DNA fragment 6, and other steps are unchanged.

7. Each single colony was streaked on LB solid medium and LB solid medium containing 34. Mu.g/mL chloramphenicol, respectively, cultured overnight at 37℃and the single colony grown on LB solid medium and not grown on LB solid medium containing 34. Mu.g/mL chloramphenicol was picked for PCR verification using primers 17 and 18, and the amplified product of the positive clone was a 1812bp fragment. 3 positive clone single colonies were picked and sent to Nanjing qing department biotechnology Co., ltd for sequencing, and the correctly sequenced single clone was preserved and named as Svp035 strain.

Example 4 construction of the tetracycline control Module (substitution of the promoter of the gltA Gene with promoter pteto)

1. The plasmid pXZ-CS was used as a template, and PCR was performed using a primer set comprising primer 19 and primer 20 (reaction system and reaction conditions were referred to in step 1 of example 1, respectively), to obtain a DNA fragment 7 having a size of 2719 bp. The DNA fragment 7 has 50bp homologous region with the upstream and downstream of the promoter of the gltA gene, and is used for the first step of homologous recombination and provides a rescreening mark for the second step of homologous recombination.

2. The pKD46 plasmid was transformed into competent cells of the Svp035 strain to give recombinant Svp035-pKD46.

3. The DNA fragment 7 was transformed into recombinant Svp035-pKD46 in the same manner as in step 3 of example 1 to obtain Svp036 strain. Specifically, the competent cells of the recombinant bacteria Svp024A-pKD46 in the step 3 in the example 1 are replaced by competent cells of the recombinant bacteria Svp035-pKD46, the DNA fragment 1 is replaced by the DNA fragment 7, and other steps are unchanged, so that single colony is obtained. Single colonies were selected for PCR verification using primers 25 and 26, and the amplified product of the positive clone was a 3856bp fragment. 3 positive clone single colonies were picked and sent to Nanjing qing department biotechnology Co., ltd for sequencing, and the correctly sequenced single clone was preserved and named as Svp036 strain.

4. PCR amplification was performed using the genomic DNA of the Svp001 strain as a template and a primer set consisting of primer 21 and primer 22 (reaction system and reaction conditions were respectively referred to the reaction system and reaction conditions in step 1 of example 1), to obtain a DNA fragment 8 having a size of 547 bp. PCR amplification was performed using the genomic DNA of the Svp001 strain as a template and a primer pair consisting of the primer 23 and the primer 24 (the reaction system and the reaction conditions were referred to in step 1 of example 1, respectively), to obtain a DNA fragment 9 having a size of 505 bp. The DNA fragment 8 and the DNA fragment 9 were subjected to product purification, then mixed and used as templates, and PCR amplification was performed using a primer pair consisting of the primer 21 and the primer 24 (the reaction system and the reaction conditions were respectively referred to the reaction system and the reaction conditions in step 1 of example 1), to obtain the DNA fragment 10 having the nucleotide sequence shown in SEQ ID NO. 4.

SEQ ID NO. 4 is promoter pteto at position 509-547 from the 5' end, ptetO is blocked from regulation by the repressor gene tetR.

5. The pKD46 plasmid was transformed into competent cells of Svp036 strain to obtain recombinant Svp036-pKD46.

6. The DNA fragment 10 was electrotransferred into recombinant Svp036-pKD46 as in step 6 of example 1 to give a single colony. Specifically, the competent cells of the recombinant bacteria Svp030-pKD46 are replaced by competent cells of the recombinant bacteria Svp036-pKD46, the DNA fragment 2 is replaced by the DNA fragment 10, and other steps are unchanged.

7. Each single colony was streaked on LB solid medium and LB solid medium containing 34 μg/mL chloramphenicol, cultured overnight at 37 ℃, and the single colony grown on LB solid medium but not on LB solid medium containing 34 μg/mL chloramphenicol was picked for PCR verification, primers used as primer 25 and primer 26, the amplified product of the positive clone was a 1276bp fragment, 3 positive clone single colonies were picked and sent to nanjing qing biotechnology limited for sequencing, and the correctly sequenced single clone was preserved and designated as Svp037 strain.

Example 5 random mutagenesis based on promoter pLux

1. The plasmid pXZ-CS was used as a template, and PCR was performed using a primer set consisting of the primer 27 and the primer 28 (reaction system and reaction conditions were respectively referred to the reaction system and reaction conditions in step 1 of example 1), to obtain a DNA fragment 11 having a size of 2719 bp. The DNA fragment 11 has 50bp homologous region on the upstream and downstream of pLux-tetR gene, and is used for first step homologous recombination and provides a rescreen marker for second step homologous recombination.

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

3. The DNA fragment 11 was transformed into recombinant Svp037-pKD46 in the same manner as in step 3 of example 1 to obtain Svp038 strain. Specifically, the competent cells of the recombinant bacteria Svp024A-pKD46 in the step 3 in the example 1 are replaced by competent cells of the recombinant bacteria Svp037-pKD46, the DNA fragment 1 is replaced by the DNA fragment 11, and other steps are unchanged, so that single colony is obtained. Single colonies were selected for PCR verification using primers 17 and 18, and the amplified product of the positive clone was a 4431bp fragment. 3 positive clone single colonies were picked and sent to Nanjing qing department biotechnology Co., ltd for sequencing, and the correctly sequenced single clone was preserved and named Svp038 strain.

4. And (3) taking the genome of the Svp035 strain as a template, and carrying out PCR (polymerase chain reaction) amplification by adopting a primer pair consisting of a primer 29 and a primer 30 to obtain an amplified fragment pLux ¹.

According to the above method, the primer 30 is replaced with the primer 31 to the primer 44, respectively, and the other steps are unchanged, thereby obtaining the amplified fragment pLux ² to the amplified fragment pLux ¹⁵ in sequence.

The nucleotide sequence of amplified fragment pLux ¹ -amplified fragment pLux ¹⁵ is shown in Table 3.

The 15 primer pairs mutate and amplify the interval region of the promoter pLux respectively, while the conserved region is reserved.

5. The pKD46 plasmid was transformed into competent cells of Svp038 strain to obtain recombinant Svp038-pKD46.

6. The amplified fragment pLux ¹ was electrotransferred into recombinant Svp038-pKD46 as in step 6 of example 1, resulting in a single colony. Specifically, the competent cells of the recombinant bacteria Svp030-pKD46 are replaced by competent cells of the recombinant bacteria Svp038-pKD46, the DNA fragment 2 is replaced by an amplified fragment pLux ¹, and other steps are unchanged.

7. Each single colony was streaked on LB solid medium and LB solid medium containing 34 μg/mL chloramphenicol, cultured overnight at 37 ℃, and the single colony grown on LB solid medium but not on LB solid medium containing 34 μg/mL chloramphenicol was picked for PCR verification, primers used as primer 17 and primer 18, amplified product of positive clone was 1812bp fragment, 3 positive clone single colonies were picked and sent to nanjing qing biotechnology limited for sequencing, and the correctly sequenced single clone was preserved and designated as Svp039 strain.

According to the methods of the above steps 6 and 7, the amplified fragment pLux ¹ is replaced with the amplified fragment pLux ² -amplified fragment pLux ¹⁵, respectively, and the other steps are unchanged, so that the Svp040 strain, the Svp041 strain, the Svp042 strain, the Svp043 strain, the Svp044 strain, the Svp045 strain, the Svp046 strain, the Svp047 strain, the Svp048 strain, the Svp049 strain, the Svp050 strain, the Svp051 strain, the Svp052 strain and the Svp053 strain are sequentially obtained.

EXAMPLE 6 production of pantoic acid by high Density fermentation of Svp024A Strain, svp037 Strain, svp039 Strain-Svp 053 Strain

1. High density fermentation

(1) The strain to be tested (Svp 024A 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 to LB solid culture medium for activation, the strain to be tested is selected and inoculated to 3ml LB liquid culture medium in a single clone mode, and the culture is carried out at 37 ℃ and 200rpm overnight to obtain primary seed culture liquid.

(2) The primary seed culture solution was inoculated into 50ml of a synthetic medium at an inoculum size of 5% (v/v), and cultured at 37℃and 200rpm for 6-7 hours to obtain a secondary seed culture solution.

The solute of the synthetic culture medium has the concentration of 2.63g/L of diammonium phosphate, 0.87g/L of monoammonium phosphate, 0.37g/L of potassium chloride, 0.37g/L of magnesium sulfate heptahydrate, 0.154g/L, AM1 of trace metal salt, 1.5mL/L of glucose monohydrate and the solvent of water.

(3) The secondary seed culture solution was inoculated into a fermenter (specification: 5L) containing 2000ml of synthetic medium, and fermentation was performed for 72 hours, and the fermentation solution was taken every 4 hours from the point of rebound of dissolved oxygen.

The set values of all parameters in the fermentation process are 37 ℃ and pH7.0, dissolved oxygen is 30%, ventilation flow is 1.5vvm, stirring speed is 300-1000 r/min, and the dissolved oxygen, stirring speed and ventilation are cascaded.

The feeding strategy is that when the dissolved oxygen value is more than 60%, feeding culture medium is added into the fermentation tank, and when the dissolved oxygen value is less than 60%, feeding is stopped.

The solute of the feed medium and the concentration thereof are 75% (m/v) glucose and 6g/L monopotassium phosphate, and the solvent is water.

2. Fermentation broth detection

The fermentation broths collected in the step 1 at different time points are detected as follows:

(1) The fermentation broth was taken and checked for OD _550nm.

(2) Taking 1mL of fermentation liquor, centrifuging 10000g for 1min, and collecting supernatant.

(3) The supernatant collected in the step (2) is diluted by 20 times with sterile water, and then filtered by a 0.22 mu m filter membrane, and the filtrate is collected.

(4) And (3) detecting the filtrate collected in the step (3) by adopting High Performance Liquid Chromatography (HPLC) to obtain the content of pantoic acid in the fermentation liquor.

The High Performance Liquid Chromatography (HPLC) detection conditions are that the chromatographic column is GL SciencesWondasilC chromatographic columns (4.6X105 mm;5 μm), the mobile phase is 0.1% phosphoric acid aqueous solution, acetonitrile=19:1, the wavelength is 210nm, the column temperature is 35 ℃, the flow rate is 1.0mL/min, and the sample injection amount is 10. Mu.L.

The partial detection results within 72h of fermentation are shown in FIG. 1 (strain Svp 024A), FIG. 2 (strain Svp 037) and FIG. 3 (strain Svp 045).

The result shows that the biomass of the Svp024A strain can reach 123.2, when the biomass of the Svp037 strain after the quorum sensing element is introduced reaches about 10 at OD _550nm, the quorum sensing element Lux system is triggered immediately, the signal molecule AHL triggers pLux promoter to express the repressor tetR to repress the expression of the gltA gene, so that the tricarboxylic acid cycle of the strain can not normally circulate, and the growth of the strain is only about 10, which directly influences the growth of the strain, so that the strain can not continue to ferment in a 5L tank, and the pantoic acid also can not begin to accumulate. The inventors speculate that promoter pLux and signaling molecule AHL responses are too sensitive, triggering quorum sensing at very low thresholds.

In order to break through the technical bottleneck, the inventor of the application reasonably designs and synthesizes 15 mutants (namely promoter pLux ¹～pLux¹⁵) of a promoter pLux through a large number of experimental analysis, then sequentially obtains 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 for expressing the pLux mutants through gene editing, and finally ferments the strains. The fermentation result shows that the Svp045 strain responds when the biomass is between OD _550nm and 30-60, the growth of thalli is slowed down until the biomass is reduced, the corresponding pyruvic acid enters into the metabolic flow of the pantoic acid to start accumulating the product pantoic acid, and finally the yield of the pantoic acid reaches 26.11g/L, at the moment, the OD _550nm of the Svp045 strain is only 45, and meanwhile, compared with the starting strain (namely the Svp024A strain, the highest biomass is 123.2), the biomass of the Svp045 strain is 72 at most, and the biomass is reduced by more than 50%.

Therefore, the quorum sensing element has obvious positive effects in the aspect of controlling biomass accumulation of the pantoic acid-producing microorganisms, and after reasonable modification of the promoter pLux (particularly the promoter pLux ⁷, the nucleotide sequence of which is shown as SEQ ID NO: 5), the quorum sensing element meets the requirement of industrial production, so that not only is the excessive biomass accumulation of the pantoic acid-producing engineering bacteria in the industrial production solved, but also the yield of pantoic acid is improved, and a foundation is laid for realizing the fermentation production of pantoic acid by the industrial microorganisms.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims (10)

1. A pLux promoter has the nucleotide sequence 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 and control system comprises a LuxI module, a LuxR module, a tetR module and a tetracycline regulation and control module;

the LuxI module comprises a gene for encoding the LuxI protein and an expression element I for expressing the LuxI protein;

The LuxR module comprises a gene for encoding the LuxR protein and an expression element II for expressing the LuxR protein;

The tetR module comprises a gene encoding a repressor tetR and an expression element III for expressing the repressor tetR;

The tetracycline regulation module comprises an expression element IV for down-regulating the expression of the citrate synthase gltA;

the expression element I and the expression element II are constitutive strong promoters, and the expression element III and the expression element IV are induction elements;

preferably, the first expression element is a constitutive strong promoter M1-93, the second expression element is a constitutive strong promoter lpp, the third expression element is a promoter pLux, and the fourth expression element is a promoter Pteto;

More preferably, the nucleotide sequence of promoter pLux is shown in SEQ ID NO. 5.

4. A recombinant bacterium for producing pantoic acid is used for expressing LuxI protein, luxR protein and repressor protein tetR in a starting strain body and down-regulating the expression of citric acid synthase gltA, wherein the repressor protein tetR is used for promoting the expression under the control of a promoter pLux, and the citric acid synthase gltA is down-regulated under the control of the promoter Pteto;

Preferably, the nucleotide sequence of the promoter pLux is shown in SEQ ID NO. 5.

5. The recombinant bacterium producing pantoic acid according to claim 4, wherein the starting strain is E.coli which weakens ilvE gene encoding branched-chain amino acid transaminase, knocks out avtA gene encoding valine pyruvic acid aminotransferase, knocks out leuDH gene encoding amino acid dehydrogenase, knocks out mlaZ gene encoding maltodextrin glucosidase, knocks out tdcB gene encoding threonine dehydratase and overexpresses panB gene encoding ketopantothenate hydroxymethyltransferase and panE gene encoding ketopantoic acid reductase.

6. A preparation method of a recombinant bacterium producing pantoic acid comprises the following steps of introducing a gene encoding LuxI protein, a gene encoding LuxR protein and a gene encoding a repressor tetR into a starting strain, regulating the gene encoding the LuxI protein by using promoters M1-93 to improve the expression quantity of the LuxI protein, regulating the gene encoding the repressor tetR by using a promoter pLux to improve the expression quantity of the repressor tetR, regulating the gene encoding the LuxR protein by using a promoter lpp to improve the expression quantity of the LuxR protein, regulating the gene encoding the citrate synthase gltA by using a promoter Pteto to reduce the expression quantity of the citrate synthase gltA, wherein 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 method according to claim 6, wherein the starting strain is Escherichia coli modified by weakening ilvE gene encoding branched-chain amino acid transaminase, knocking out avtA gene encoding valine pyruvic aminotransferase, knocking out leuDH gene encoding amino acid dehydrogenase, knocking out mlaZ gene encoding maltodextrin glucosidase, knocking out tdcB gene encoding threonine dehydratase, and overexpressing panB gene encoding ketopantothenate hydroxymethyltransferase and panE gene encoding ketopantoate reductase.

8. The method according to claim 7, wherein said modification of said starting strain further comprises at least one of knocking out the sdaA gene encoding L-serine deaminase I, knocking out the sdaB gene encoding L-serine deaminase II, and knocking out the panF gene encoding pantothenate permease.

9. A process for producing pantoic acid, which comprises the steps of fermenting and culturing the recombinant bacterium for producing pantoic acid according to claim 4 or 5 or the recombinant bacterium for producing pantoic acid prepared by the process according to any one of 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) to A6);

Or, the use of the dynamic control system of claim 3 in any one of the following A2) -A6);

or, the recombinant bacterium for producing pantoic acid according to claim 4 or 5, wherein the recombinant bacterium is used in any one of the following A3) to A5);

Or, the use of the recombinant bacteria producing pantoic acid prepared by the preparation method of any one of claims 6 to 8 in any one of the following A3) to A5);

or, the use of the process for producing pantoic acid according to claim 9 in any one of the following A4) to A6):

A1 A dynamic regulation system is constructed;

A2 Constructing recombinant bacteria for producing pantoic acid;

a3 Producing pantoic acid;

a4 Improving the yield of pantoic acid;

A5 Reducing the biomass of the strain used to produce pantoic acid;

a6 Increased production of pantoic acid and decreased biomass of the strain used to produce pantoic acid.