A quaternary pyrimidine gene cluster derived from halophilic bacillus, mutants and applications

Abstract

The invention discloses an ectoine gene cluster, mutant and application derived from halophilic bacillus, belonging to the field of synthetic biotechnology. The ectoine gene cluster nucleic acid sequence is shown in SEQ ID NO.7, including the following three genes: ectA gene, the nucleic acid sequence is shown in SEQ ID NO.1, ectB gene, the nucleic acid sequence is shown in SEQ ID NO.2, ectC gene, the nucleic acid sequence is shown in SEQ ID NO.3, and the gene cluster is designed and mutated to obtain a mutant, the nucleic acid sequence is shown in SEQ ID NO.8. The invention can improve the synthesis efficiency of ectoine, improve the fermentation content of ectoine, reduce the production cost of ectoine, and is conducive to the popularization and application of ectoine by constructing a recombinant strain using the ectoine gene cluster and its mutant.

Description

Technical Field

[0001] The invention relates to an ectoine gene cluster, a mutant and an application derived from halophilic bacillus, and belongs to the field of synthetic biotechnology. Background Art

[0002] Tetrahydropyrimidine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid, C6H 10 N2O2,M W =142.16) is a cyclic amino acid derivative. It was first discovered in the photosynthetic purple bacterium Halochloris of the extremely halophilic genus Ectothiorhodspira. It was subsequently found to be commonly found in halophilic or salt-tolerant microorganisms. It has a strong ability to complex water molecules and has a good protective effect on proteins, nucleic acids, enzymes, biofilms and cells under adverse conditions such as high osmotic pressure, high temperature, low temperature, dryness, strong acid and alkali, and radiation. It is currently widely used in the fields of medicine, cosmetics, and biological preparations, and has broad prospects.

[0003] There are two main methods for the synthesis of tetrahydropyrimidine: chemical method and biological method. The biological synthesis method is widely used because of its simple production process, high synthesis rate, low difficulty in subsequent separation and purification, and environmental friendliness compared to the chemical synthesis method. The biosynthesis of ectoine is divided into three steps: first, L-aspartate-β-semialdehyde (ASA) is used as a substrate, catalyzed by 2,4-diaminobutyric acid aminotransferase (EctB) to produce L-2,4-diaminobutyric acid (DABA); second, under the catalysis of 2,4-diaminobutyric acid acetyltransferase (EctA), DABA is acetylated to produce N-acetyl-L-2,4-diaminobutyric acid (ADABA); finally, ectoine synthase (EctC) cyclizes ADABA to form ectoine.

[0004] In the early stage, the biosynthesis of ectoine was mostly produced by the "bacterial milking" method of halomonas, that is, cells were first cultured under high osmotic pressure to accumulate ectoine inside the cells, and then hypotonic shock was used to stimulate the release of ectoine from the cells to the extracellular space, and then the hypertonic culture and hypotonic shock were repeated to obtain a higher concentration of ectoine. This process is complicated and requires cultivation under high salt conditions, so it has high requirements for equipment, and the discharge of high-salt waste liquid will cause environmental problems, resulting in its production price being very expensive.

[0005] In recent years, researchers have constructed engineered strains by heterologously expressing ectoine gene clusters in Escherichia coli and Corynebacterium glutamicum, which has reduced the difficulty of fermentation and increased the fermentation content of ectoine. However, the ectoine gene clusters used to construct the engineered strains are mostly derived from Halomonas, such as H.elongate DSM 2581, and the gene cluster sequences are highly conserved. Therefore, developing new ectoine gene resources, increasing the fermentation content of ectoine, reducing its production cost, and improving production efficiency are of great practical significance for the application of ectoine. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide an ectoine gene cluster, mutants and applications derived from halophilic Bacillus. By using the ectoine gene cluster and mutants derived from halophilic Bacillus to construct a recombinant ectoine engineering strain, the ectoine content obtained by fermentation is high.

[0007] In order to solve the above technical problems, the technical solutions provided by the present invention are as follows:

[0008] The present invention provides an ectoine gene cluster derived from halophilic bacillus, the ectoine gene cluster nucleic acid sequence is shown in SEQ ID NO.7, and includes the following three genes: ectA gene, the nucleic acid sequence is shown in SEQ ID NO.1; ectB gene, the nucleic acid sequence is shown in SEQ ID NO.2; ectC gene, the nucleic acid sequence is shown in SEQ ID NO.3. The ectA, ectB, ectC gene sequences of the ectoine gene cluster are all different from the gene sequences in the database, and are a new ectoine synthesis gene sequence.

[0009] Furthermore, the ectoine gene cluster is derived from Halobacillus sp. FL-2423, which has the characteristic of ectoine secretion. The strain was deposited in the General Microbiology Center of China Microorganism Culture Collection on November 1, 2019, with the deposit number CGMCC NO.18783, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The present invention also provides a mutant formed by an ectoine gene cluster derived from halophilic Bacillus, and the nucleic acid sequence of the mutant is shown in SEQ ID NO.8.

[0011] Furthermore, the mutant consists of the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.

[0012] Furthermore, the amino acid sequence shown in SEQ ID NO.4 is a nucleic acid sequence such as the original sequence of the ectA gene shown in SEQ ID NO.1, and has the following mutations: the polypeptide amino acid sequence corresponding to the nucleic acid sequence is replaced by L to I at the 54th amino acid, and by Q to T at the 103rd amino acid.

[0013] Furthermore, the amino acid sequence shown in SEQ ID NO.5 is a nucleic acid sequence such as the original sequence of the ectB gene shown in SEQ ID NO.2, and has the following mutations: the polypeptide amino acid sequence corresponding to the nucleic acid sequence is replaced by R with Q at the 28th amino acid, the 67th amino acid is replaced by L with P, the 189th amino acid is replaced by L with T, the 251st amino acid is replaced by F with H, and the 331st amino acid is replaced by V with E.

[0014] Furthermore, the amino acid sequence shown in SEQ ID NO.6 is a nucleic acid sequence such as the original sequence of the ectC gene shown in SEQ ID NO.3, with the following mutations: the polypeptide amino acid sequence corresponding to the nucleic acid sequence is replaced by A with E at the 46th amino acid, by D with R at the 90th amino acid, and by Q with E at the 134th amino acid.

[0015] The present invention further provides an application of an ectoine gene cluster derived from halophilic Bacillus and a mutant formed therefrom in a process of synthesizing ectoine, comprising the following steps:

[0016] (1) Obtaining the tetrahydropyrimidine gene cluster of Halobacillus FL-2423;

[0017] (2) Preparation of ectoine gene cluster mutants of Halobacillus sp. FL-2423;

[0018] (3) constructing a recombinant vector containing the FL-2423 ectoine gene cluster or an ectoine gene cluster mutant;

[0019] (4) Construction of ectoine recombinant strains;

[0020] (5) Fermentation to produce tetrahydropyrimidine using glucose as substrate.

[0021] Note: IPTG is isopropyl-β-D-thiogalactoside.

[0022] Furthermore, the specific operation of step (1) is:

[0023] (1-1) The activated FL-2423 strain was inoculated into a high-salt culture medium and cultured at 28°C for 16 h. The genomic DNA was extracted using a bacterial genome extraction kit and sent to Qingke Bio for genome sequencing;

[0024] (1-2) Search the nucleic acid and amino acid sequences of the Halomonas elongata DSM 2581 ectoine gene ectA (GenBank record: CBV42472.1), ectB (GenBank record: CBV42473.1), and ectC (GenBank record: CBV42474.1) from the NCBI database;

[0025] (1-3) The nucleic acid and amino acid sequences of ectA, ectB, and ectC of DSM 2581 were compared with the genome sequencing results and gene structure annotation results of FL-2423 for homology analysis to obtain the nucleic acid sequences of ectA, ectB, and ectC of FL-2423;

[0026] (1-4) The ectA, ectB, and ectC nucleic acid sequences of FL-2423 were analyzed by phylogenetic tree analysis using NCBINucleotide BLAST in the NCBI database. The results are shown in the attached figure. Figure 1 , 2 , 3;

[0027] Furthermore, in step (1-1), the components of the high-salt culture medium are 30.0 g / L sodium chloride, 10.0 g / L glucose, 1.0 g / L yeast powder, 2.5 g / L peptone, 2.0 g / L disodium hydrogen phosphate, 2.0 g / L sodium dihydrogen phosphate, 0.85 g / L potassium chloride, 0.4 g / L magnesium sulfate, 0.01 g / L manganese sulfate, 0.01 g / L ferrous sulfate, 20.0 g / L calcium carbonate, and the pH is 7.0.

[0028] Furthermore, the specific operation of step (2) is as follows: using the genome of FL-2423 as a template, using primers 2423ectABC-F / ectABC-R, amplifying the ectoine gene cluster to obtain the FL-2423-ectABC fragment, performing site-directed mutagenesis design on FL-2423-ectABC, and synthesizing the mutated FL-2423-ectABC mut Fragment.

[0029] Furthermore, the specific operation of step (3) is: using the plasmid as a template, using primers to amplify the obtained linearized fragment; using FL-2423-ectABC or FL-2423-ectABC mut As a template, FL-2423-ectABC or FL-2423-ectABC containing the vector homology arm was amplified. mut The ectoine gene cluster was connected with the linearized fragment and the ectoine gene cluster using homologous recombinase, and transformed into Escherichia coli DH5α. After resistance screening, the plasmid was extracted to obtain a recombinant expression vector.

[0030] Furthermore, in step (3), the resistance screening is ampicillin resistance screening or kanamycin resistance screening.

[0031] Furthermore, the specific operation of step (4) is: transforming the expression vector into competent cells of Escherichia coli BL21 (DE3) by heat shock method to obtain a recombinant strain, wherein the temperature of the heat shock method is 42° C. and the time is 90 s.

[0032] Furthermore, step (5) step (5) includes seed plate activation, seed liquid preparation, and fermentation to produce ectoine, and the specific operations are:

[0033] (5-1) Plate seed activation: Streak the ectoine recombinant strain on an eggplant-shaped bottle containing solid LB medium containing resistance in a clean bench and culture at 37°C for 12 h to obtain activated seeds;

[0034] (5-2) Seed solution preparation: The activated seeds in the eggplant-shaped bottle were placed in a fermentation tank containing 10 L of seed culture medium at 37°C, with a dissolved oxygen content of 20-30%. The pH was adjusted to 7.0 with ammonia water, and the culture was continued until the OD reached 15 to obtain seed solution.

[0035] (5-3) Fermentation to produce tetrahydropyrimidine: The seed liquid was inoculated into a fermentation tank containing 8 L of fermentation medium at 37 °C and 20-30% dissolved oxygen. The initial pH was adjusted to 7.0 with ammonia water. After the substrate sugar was consumed, IPTG was added to the fermentation medium to a concentration of 0.1 mM / L. A glucose solution with a concentration of 700 g / L was added. The growth rate of OD was adjusted to 2-6 / h. Ammonia water was added to adjust the pH to 7.4. When the OD grew to 55, the sugar addition rate was accelerated to a sugar concentration of 0.2-1 g / L. The fermentation was completed after 30 h to obtain a fermentation liquid containing tetrahydropyrimidine.

[0036] Furthermore, in step (5-2), the components of the seed culture medium are: 30.0 g / L glucose, 1.5 g / L peptone, 6.8 g / L yeast powder, 0.68 g / L citric acid monohydrate, 1.6 g / L potassium dihydrogen phosphate, 0.68 g / L magnesium sulfate heptahydrate, and 0.33 g / L GPE defoamer.

[0037] Furthermore, in step (5-3), the seed liquid accounts for 15% of the fermentation medium by volume.

[0038] Further, in step (5-3), the composition of the fermentation medium is: glucose 25.0g / L, peptone 2.5g / L, yeast powder 3.8g / L, corn steep liquor dry powder 6.8g / L, citric acid monohydrate 2.5g / L, potassium dihydrogen phosphate 3.8g / L, magnesium sulfate heptahydrate 1.5g / L, GPE defoamer 0.85g / L. When the glucose content in the fermentation broth is high, even if an inducer is present, it will have an adverse effect on the induction and is not conducive to the expression of the enzyme. Therefore, generally, fermentation requires induction after the substrate sugar is consumed. The substrate sugar concentration is crucial to the synthesis of the enzyme that needs to be induced to express. In addition, the addition of an inducer will inhibit cell growth. The initial low glucose concentration will result in a small OD during induction. The low biomass during induction will cause the bacteria to be unable to grow to a higher cell concentration, resulting in a low content of tetrahydropyrimidine; the initial high glucose concentration, the biomass is too high after the substrate sugar is consumed, the bacteria are in an aging state, the vitality decreases, the induction effect decreases, and the content of tetrahydropyrimidine is also low.

[0039] Furthermore, in step (5-3), after the substrate sugar is consumed, IPTG is added to the fermentation medium to a concentration of 0.1 mM / L, and a glucose solution with a concentration of 700 g / L is fed to adjust the OD growth rate to 2-4 / h. After adding the inducer, the strain begins to transform from reproductive growth to the synthesis of ectoine-related enzymes, and the OD growth rate is controlled by feeding glucose. When the OD grows too slowly, the inducer causes greater damage to the cells, resulting in a lower final biomass and a low ectoine content; when the OD grows too fast, the induction effect is poor, the expression of ectoine-related enzymes is low, and the fermentation content of ectoine is low.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1. The ectoine gene cluster provided by the present invention is derived from Halobacillus sp. FL-2423, which has the characteristic of secreting ectoine. The NCBI Nucleotide BLAST analysis shows that the ectoine gene cluster is different from the existing ectoine gene coding sequences and is a new ectoine synthesis gene sequence.

[0042] 2. The ectoine gene cluster of FL-2423 provided by the present invention has a better efficiency in producing ectoine by the enzyme catalyzed by the enzyme than the commonly used ectoine gene cluster of Halomonas elongata DSM 2581. Under the same fermentation conditions, the ectoine content is increased by more than 20%.

[0043] 3. The present invention carries out mutation design on the ectoine gene cluster derived from halophilic Bacillus to obtain a mutant formed by the ectoine gene cluster, which has enhanced activity and greatly improved ectoine synthesis efficiency. Under the same fermentation conditions, compared with the strain constructed by the original ectoine gene cluster of Halobacillus sp. FL-2423, the fermentation ectoine content of the mutant formed by the ectoine gene cluster is increased by more than 30%; compared with the strain constructed by the ectoine gene cluster of Halomonas elongata DSM 2581, the fermentation content of ectoine of the mutant recombinant strain is increased by more than 60%.

[0044] 4. The ectoine gene cluster and mutants derived from Bacillus halophilus were applied to improve the synthesis efficiency of ectoine, thereby increasing the fermentation content of ectoine and reducing the production cost of ectoine, which is conducive to the promotion and application of ectoine. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is the phylogenetic tree analysis of ectA gene of FL-2423;

[0046] Figure 2 It is the phylogenetic tree analysis of ectB gene of FL-2423;

[0047] Figure 3 This is the phylogenetic tree analysis of the ectC gene of FL-2423. DETAILED DESCRIPTION

[0048] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. At the same time, the experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents, materials, and instruments are all prior art and can be obtained from commercial channels unless otherwise specified.

[0049] Example 1 Obtaining the ectoine gene cluster of Halobacillus sp. FL-2423

[0050] (1) The activated FL-2423 strain was inoculated into a high-salt culture medium and cultured at 28°C for 16 h. The genomic DNA was extracted using a bacterial genome extraction kit and sent to Qingke Biotechnology for genome sequencing and gene structure annotation. The components of the high-salt culture medium were: sodium chloride 30.0 g / L, glucose 10.0 g / L, yeast powder 1.0 g / L, peptone 2.5 g / L, disodium hydrogen phosphate 2.0 g / L, sodium dihydrogen phosphate 2.0 g / L, potassium chloride 0.85 g / L, magnesium sulfate 0.4 g / L, manganese sulfate 0.01 g / L, ferrous sulfate 0.01 g / L, calcium carbonate 20.0 g / L, pH 7.0;

[0051] (2) Search the nucleotide and amino acid sequences of the Halomonas elongata DSM 2581 ectoine gene ectA (GenBank record: CBV42472.1), ectB (GenBank record: CBV42473.1), and ectC (GenBank record: CBV42474.1) from the NCBI database;

[0052] (3) The nucleic acid sequences and amino acid sequences of ectA, ectB, and ectC of DSM 2581 were compared with the genome sequencing results and gene structure annotation results of FL-2423 for homology analysis, and the nucleic acid sequences of ectA, ectB, and ectC of FL-2423 were obtained, as shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3;

[0053] (4) The ectA, ectB, and ectC nucleic acid sequences of FL-2423 were analyzed by phylogenetic tree analysis using NCBINucleotide BLAST in the NCBI database. The results are shown in the attached figure. Figure 1 , 2 、3.

[0054] SEQ ID NO.1: FL-2423-ectA

[0055] atgaacgtgaccacagagcccttcattccctccgccgagctcgcccggcccagcattgccgatgccgtggtcggccatcaggccacgccgctgttcatccgcaagcccagccccgacgacggctggggcatctacgaactggtcaagtcctgcccgccgctggacgtcaattccgcctatgcctacctgctgctggccacccagttccgcgacagctgtgccgtggctaccaacgaggagggcgagatcgtcggcttcgtttccggctacgtgaagagcaactgcccggacacctatttcctctggcaggtcgcggtgggtgagaaggcgcgcggcaccggcctggcccgccgcctggtcgaagccgtgatgacccgcccggagatggccgaggtgcaccacctcgagaccaccatcactcccgacaaccaggcatcctgggggctcttccggcgcctcgccgaccgctggcaggcgccgctcaacagccgcgagtacttctccaccgaccagctcggcggcgagcatgacccggaaaacctcgtccgcatcggtcccttccagaccgaccagatctga

[0056] SEQ ID NO.2:FL-2423-ectB

[0057]

[0058] SEQ ID NO.3: FL-2423-ectC

[0059] atgatcgttcgcaacctcgaagtcgcccgcgagaccgaccgtctggtcaccgccgaaaacggcaactgggacagcacccgcctgtctctggccgaagatggtg gcaactgctccttccacatcacccgtatctatgccggcaccgagacccatatccactacaagcatcacttcgaggcggtttattgcatcgaaggcgagggcgaa gtggaaaccctggccgatggcaagatctggccgatcaagccgggcgacatctatatcctcgaccagcacgacgagcacctgctgcgggcgtacaagaccatgc acctggcctgcgtgttcacgccgggcctgaccggcaacgaggtgcaccgcgaagacggttcctacgcacctgccgacgaagccgacgaccagaagccgctgtaa

[0060] Example 2 Preparation of Halobacillus sp. FL-2423 Tetrahydropyrimidine Gene Cluster Mutants

[0061] The genome of FL-2423 was used as a template, and primers 2423ectABC-F / ectABC-R were used to amplify the ectoine gene cluster to obtain the FL-2423-ectABC fragment, the nucleic acid sequence of which is shown in SEQ ID NO.7. Site-directed mutagenesis of ectABC was designed, and the mutant FL-2423-ectABC was synthesized. mut The fragment, the nucleic acid sequence is shown as SEQ ID NO.8, and the amino acid sequence is shown as SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6.

[0062] SEQ ID NO.4

[0063] MNVTTEPFIPSAELARPSIADAVVGHQATPLFIRKPSPDDGWGIYELVKSCPPIDVNSAYAYLLLATQFRDSCAVATNEEGEIVGFVSGYVKSNCPDTYFLWTVAVGEKARGTGLARRLVEAVMTRPEMAEVHHLETTITPDNQASWGLFRRLADRWQAPLNSREYFSTDQLGGEHDPENLVRIGPFQTDQI*

[0064] SEQ ID NO.5

[0065] MQTQILERMESEVRTYSRSFPVVFTKAQNARLTDEDGREYIDFLAGAGTLNYGHNNPKIKQALVDYPASDGIVHGLDFWTAAKRDYLETLEEVILKPRGLDYKVHLPGPTGTNAVEAAIRLARVAKGRHNIVTFTNGFHGVTMGALATTGNRKFREATGGIPTQGASFLPYDGYMGEHTDTLDYFEKLTGDKSGGLDLPAAVIVETVQGEGGINVAGLDWLKRLEGICRAHDILLIVDDIPAGCGRTGKFHSFEHAGVTRDIVTNSKSLSGYGLPFAHVLMRPELDKWKPGQYNGTFRGFNLAFTTAAATLRQYWSDDVFERDVQRKGRVEADRFQKIAAWLSENGIEASERGRGLMRGIDVGSGDIADKITHQAFENGLVIETSGQDGEVVKCLCPLTIPDEDLIEALDILEASARQALS*

[0066] SEQ ID NO.6

[0067] MIVRNLEVARETDRLVTAENGNWDSTRLSLAEDGGNCSFHITRIYEGTETHIHYKHHFEAVYCIEGEGEVETLADGKIWPIKPGDIYILRQHDEHLLRAYKTMHLACVFTPGLTGNEVHREDGSYAPADEADDEKPL*

[0068] SEQ ID NO.7: Halobacillus sp. FL-2423 ectoine gene cluster

[0069]

[0070] SEQ ID NO.8: Halobacillus sp. FL-2423 tetrahydropyrimidine gene cluster mutant

[0071]

[0072] Example 3 Construction of recombinant strain BL21(DE3) / pET-15b-2423ectABC

[0073] (1) Construction of recombinant expression vector

[0074] Using pET-15b plasmid as a template, primers 15b-F / R were used to amplify the linearized fragment of pET-15b; using FL-2423 genomic DNA as a template, primers 15b-2423ectABC-F / R were used to amplify the ectoine gene cluster of FL-2423 containing the homologous arms of the pET-15b vector, the linearized fragment of pET-15b and the ectoine gene cluster of FL-2423 were connected with homologous recombinase, transformed into Escherichia coli DH5α, screened for ampicillin resistance, extracted the plasmid, and obtained the recombinant expression vector pET-15b-2423ectABC.

[0075] (2) Construction of ectoine recombinant strain

[0076] The recombinant expression vector pET-15b-2423ectABC was transformed into Escherichia coli BL21(DE3) competent cells by heat shock method (42°C, 90s) to obtain the recombinant strain BL21(DE3) / pET-15b-2423ectABC.

[0077] Example 4 Construction of recombinant strain BL21(DE3) / pET-28a-2423ectABC

[0078] The construction method is the same as that of Example 3, except that the plasmid used in step (1) is pET-28a, the linearization primer is 28a-F / R, the ectoine gene cluster amplification primer is 28a-2423ectABC-F / R, and kanamycin resistance is screened.

[0079] Example 5 Construction of recombinant strain BL21(DE3) / pET-15b-2423ectABC mut

[0080] (1) Construction of recombinant expression vector

[0081] Using pET-15b plasmid as template, primers 15b-F / R were used to amplify the linearized fragment of pET-15b; FL-2423-ectABC mut As a template, use primer 15b-2423ectABC-F / R mut Amplify the ectABC containing the homology arms of the pET-15b vector mut The ectoine gene cluster of the pET-15b was connected with the homologous recombination enzyme ectABCmut The ectoine gene cluster was transformed into Escherichia coli DH5α, and the ampicillin resistance was screened. The plasmid was extracted to obtain the recombinant expression vector pET-15b-2423ectABC mut .

[0082] (2) Construction of ectoine recombinant strain

[0083] The expression vector pET-15b-2423ectABC mut The recombinant strain BL21(DE3) / pET-15b-2423ectABC was obtained by heat shock method (42°C, 90s) into competent E. coli BL21(DE3) cells. mut .

[0084] Example 6 Construction of recombinant strain BL21 (DE3) / pET-28a-2423ectABC mut

[0085] The construction method is the same as that in Example 5, except that the plasmid used in step (1) is pET-28a, the linearization primer is 28a-F / R, and the ectoine gene cluster amplification primer is 28a-2423ectABC-F / R mut , kanamycin resistance screening.

[0086] Example 7 Construction of recombinant strain BL21 (DE3) / (pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd)

[0087] (1) Construction of recombinant expression vector pETDuet-ectABC mut -lysC

[0088] Using pETDuet-1 plasmid as template, primers pETDuet-F1 / R1 were used to amplify the linearized fragment of pETDuet-1; FL-2423-ectABC mut As a template, use primers pETDuet-2423ectABC mut -F / R amplification to obtain ectABC containing the homology arms of the pETDuet-1 vector mut The linearized fragment of pETDuet-1 and ectABC were ligated with homologous recombination enzymes. mut The ectoine gene cluster was transformed into Escherichia coli DH5α, and the ampicillin resistance was screened. The plasmid was extracted to obtain the recombinant expression vector pETDuet-ectABC mut .

[0089] pETDuet-ectABC mutThe plasmid was used as a template and primers pETDuet-F2 / R2 were used to amplify pETDuet-ectABC mut The linearized fragment of pETDuet-ectABC was obtained by using the genome of Corynebacterium glutamicum CGMCC 1.1886 as a template and primers pETDuet-lysC-F1 / R1 and pETDuet-lysC-F2 / R2 to amplify the pETDuet-ectABC mut The lysC gene fragment of the vector homology arm, the nucleic acid sequence is shown in SEQ ID NO.10. Use homologous recombinase to connect pETDuet-ectABC mut The linearized fragment and the lysC gene fragment were transformed into Escherichia coli DH5α, and the ampicillin resistance was screened. The plasmid was extracted to obtain the recombinant expression vector pETDuet-ectABC mut -lysC.

[0090] (2) Construction of recombinant expression vector pACYCDuet-ppc-asd

[0091] Using pACYCDuet-1 plasmid as a template, primers pACYCDuet-F1 / R1 were used to amplify the linearized fragment of pACYCDuet-1; using BL21 (DE3) genome as a template, primers pACYCDuet-ppc-F / R were used to amplify the ppc gene containing the homologous arms of the pACYCDuet-1 vector, and the nucleic acid sequence was shown in SEQ ID NO. 11. The linearized fragment of pACYCDuet-1 and the ppc gene fragment were connected with homologous recombinase, transformed into Escherichia coli DH5α, screened for chloramphenicol resistance, and the plasmid was extracted to obtain the recombinant expression vector pACYCDuet-ppc.

[0092] Using pACYCDuet-ppc plasmid as a template, primers pACYCDuet-F2 / R2 were used to amplify the linearized fragment of pACYCDuet-ppc; using BL21 (DE3) genome as a template, primers pACYCDuet-asd-F / R were used to amplify the asd gene containing the homologous arms of the pACYCDuet-ppc vector, and the nucleic acid sequence was shown in SEQ ID NO. 12. The linearized fragment of pACYCDuet-ppc and the asd gene fragment were connected with homologous recombinase, transformed into Escherichia coli DH5α, screened for chloramphenicol resistance, and the plasmid was extracted to obtain the recombinant expression vector pACYCDuet-ppc-asd.

[0093] (3) Construction of ectoine recombinant strain

[0094] The expression vector pETDuet-ectABC mut-lysC was transformed into Escherichia coli BL21(DE3) competent cells by heat shock method (42°C, 90s) to obtain the recombinant strain BL21(DE3) / pETDuet-ectABC mut -lysC.

[0095] The expression vector pACYCDuet-ppc-asd was transformed into Escherichia coli BL21(DE3) / pETDuet-ectABC by heat shock method (42°C, 90s). mut -lysC competent cells, ampicillin and chloramphenicol resistance screening to obtain the recombinant strain BL21 (DE3) / (pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd).

[0096] SEQ ID NO.10: Corynebacterium glutamicum LysC

[0097]

[0098] SEQ ID NO.11:Escherichia coli ppc

[0099]

[0100] SEQ ID NO.12:Escherichia coli asd

[0101]

[0102] Example 8 Fermentation to produce ectoine using glucose as substrate

[0103] (1) Plate seed activation

[0104] In an ultra-clean workbench, the recombinant strain was streaked on an eggplant-shaped bottle containing solid LB medium containing resistance, and cultured at 37°C for 12 hours to obtain activated seeds.

[0105] (2) Seed solution preparation

[0106] The activated seeds in the eggplant-shaped bottle were inoculated into a fermentation tank containing 10 L of seed culture medium, the temperature was 37°C, the dissolved oxygen was 20-30%, the pH was adjusted to 7.0 with ammonia water, and the culture was continued until OD = 15 to obtain the seed solution.

[0107] The components of the seed culture medium are: glucose 30.0 g / L, peptone 1.5 g / L, yeast powder 6.8 g / L, citric acid monohydrate 0.68 g / L, potassium dihydrogen phosphate 1.6 g / L, magnesium sulfate heptahydrate 0.68 g / L, and GPE defoamer 0.33 g / L.

[0108] (3) Fermentation culture

[0109] The seed liquid was inoculated into a fermentation tank containing 8L fermentation medium at a transfer rate of 15%, the temperature was 37°C, the dissolved oxygen was 20-30%, and the pH was adjusted to 7.0 with ammonia water. After the substrate sugar was consumed, IPTG was added to the fermentation medium to a concentration of 0.1mM / L, and a glucose solution with a concentration of 700g / L was added. The growth rate of OD was adjusted to 3 / h, and the pH was adjusted to 7.4 with ammonia water. When the OD grew to 55, the sugar addition rate was accelerated to a sugar concentration of 0.5g / L. The fermentation was completed after 30h to obtain a fermentation liquid containing tetrahydropyrimidine.

[0110] The fermentation medium composition is: glucose 25.0 g / L, peptone 2.5 g / L, yeast powder 3.8 g / L, corn steep liquor powder 6.8 g / L, citric acid monohydrate 2.5 g / L, potassium dihydrogen phosphate 3.8 g / L, magnesium sulfate heptahydrate 1.5 g / L, GPE defoamer 0.85 g / L.

[0111] Comparative Example 1 Construction of recombinant strain BL21 (DE3) / pET-15b-2581ectABC

[0112] (1) Construction of recombinant expression vector

[0113] Using pET-15b plasmid as a template, primers 15b-F / R were used to amplify the linearized fragment of pET-15b; using DSM2581 genomic DNA as a template, primers 15b-2581ectABC-F / R were used to amplify the ectoine gene cluster of DSM 2581 containing the homology arms of the pET-15b vector, and the nucleic acid sequence was shown in SEQ ID NO. 8. The linearized fragment of pET-15b and the ectoine gene cluster of DSM 2581 were connected with homologous recombinase, transformed into Escherichia coli DH5α, screened for ampicillin resistance, and the plasmid was extracted to obtain the recombinant expression vector pET-15b-2581ectABC.

[0114] (2) Construction of recombinant strains

[0115] The expression vector pET-15b-2581ectABC was transformed into Escherichia coli BL21(DE3) competent cells by heat shock method (42°C, 90s) to obtain the recombinant strain BL21(DE3) / pET-15b-2581ectABC.

[0116] SEQ ID NO.9: H. elongate DSM 2581 ectoine gene cluster

[0117]

[0118] Comparative Example 2 Construction of recombinant strain BL21 (DE3) / pET-28a-2581ectABC

[0119] The construction method is the same as that of comparative example 1, except that the plasmid used in step (1) is pET-28a, the linearization primer is 28a-F / R, the ectoine gene cluster amplification primer is 28a-2581ectABC-F / R, and kanamycin resistance is screened.

[0120] The primer sequences used above are listed in Table 1.

[0121] Table 1. Primer sequences

[0122]

[0123] Experimental Example 1

[0124] The recombinant strains of Comparative Example 1, Example 3, Example 5 and Example 7 were fermented under the same conditions according to the method of Example 8 to obtain a fermentation liquid containing ectoine, and the synthesis efficiency of ectoine was measured. The results are listed in Table 2.

[0125] Table 2 Synthesis efficiency of ectoine in different gene clusters

[0126]

[0127] As can be seen from Table 2, the ectoine fermentation content of the recombinant strain of Comparative Example 1 is 5.6 g / L, the ectoine fermentation content of the recombinant strain of Example 3 is 6.8 g / L, the ectoine fermentation content of the recombinant strain of Example 5 is 9.2 g / L, and the ectoine fermentation content of the recombinant strain of Example 7 is 75.6 g / L. Compared with the recombinant strain of Comparative Example 1, the ectoine fermentation content of the recombinant strain of Example 3 is increased by 21.4%, the ectoine fermentation content of the recombinant strain of Example 5 is increased by 64.3%, and the ectoine fermentation content of the recombinant strain of Example 7 is 13.5 times that of the ectoine fermentation content of the recombinant strain of Comparative Example 1; compared with the recombinant strain of Example 3, the ectoine fermentation content of the recombinant strain of Example 5 is increased by 35.3%. It is shown that the efficiency of synthesizing ectoine of the ectoine gene cluster derived from the halophilic Bacillus provided by the present invention is more than 20% higher than that of the ectoine gene cluster of the commonly used DSM 2581 strain, and the synthesis efficiency is increased by more than 60% after mutation; the efficiency of synthesizing ectoine of the ectoine gene cluster mutant derived from the halophilic Bacillus is more than 30% higher than that of the original gene cluster.

[0128] It can be seen that the fermentation content of ectoine can be increased by fermenting ectoine using the ectoine gene cluster and mutant derived from the provided halophilic Bacillus.

[0129] Experimental Example 2

[0130] The recombinant E. coli of Comparative Example 2, Example 4, Example 6 and Example 7 were fermented under the same conditions as in Example 8 to obtain a fermentation liquid containing ectoine, and the fermentation content of ectoine was measured. The results are listed in Table 3.

[0131] Table 3 Synthesis efficiency of ectoine in different gene clusters

[0132]

[0133] As can be seen from Table 3, the ectoine fermentation content of the recombinant strain of Comparative Example 2 is 8.4 g / L, the ectoine fermentation content of the recombinant strain of Example 4 is 10.8 g / L, the ectoine fermentation content of the recombinant strain of Example 6 is 14.5 g / L, and the ectoine fermentation content of the recombinant strain of Example 7 is 75.6 g / L. Compared with the recombinant strain of Comparative Example 2, the ectoine fermentation content of the recombinant strain of Example 4 is increased by 28.6%, the ectoine fermentation content of the recombinant strain of Example 6 is increased by 72.6%, and the ectoine fermentation content of the recombinant strain of Example 7 is 9 times that of the ectoine fermentation content of the recombinant strain of Comparative Example 2. Compared with the recombinant strain of Example 4, the ectoine fermentation content of the recombinant strain of Example 6 is increased by 34.3%. It is shown that the efficiency of synthesizing ectoine of the ectoine gene cluster derived from the halophilic Bacillus provided by the present invention is more than 20% higher than that of the ectoine gene cluster of the commonly used DSM 2581 strain, and the synthesis efficiency is increased by more than 70% after mutation; the efficiency of synthesizing ectoine of the ectoine gene cluster mutant derived from the halophilic Bacillus is more than 30% higher than that of the original gene cluster.

[0134] It can be seen that the fermentation content of ectoine can be increased by fermenting ectoine using the ectoine gene cluster and mutant derived from the provided halophilic Bacillus.

[0135] Experimental Example 3

[0136] The recombinant strain BL21(DE3) / (pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd) was fermented in fermentation medium with different initial glucose concentrations, and other process conditions were the same as those in Example 8. The fermentation content of ectoine was determined, and the results are listed in Table 4.

[0137] Table 4 Effect of different initial glucose concentrations

[0138]

[0139] As shown in Table 4, when the initial glucose was 5 g / L, the OD when the substrate sugar was exhausted was 4.8, and the fermentation content was 30.8 g / L. When the initial glucose was 15 g / L, the OD when the substrate sugar was exhausted was 9.5, and the fermentation content was 54.7 g / L. When the initial glucose was 25 g / L, the OD when the substrate sugar was exhausted was 30.0, and the fermentation content was 75.6 g / L. When the initial glucose was 35 g / L, the OD when the substrate sugar was exhausted was 43.2, and the fermentation content was 57.3 g / L. When the initial glucose was 45 g / L, the OD when the substrate sugar was exhausted was 52.1, and the fermentation content was 42.6 g / L. It can be seen that when the substrate sugar concentration is 25 g / L, the fermentation content of tetrahydropyrimidine is the highest. Because adding inducers will inhibit cell growth, the initial glucose concentration is low, resulting in a small OD during induction. The low biomass during induction will cause the bacteria to be unable to grow to a higher cell concentration, resulting in a low ectoine content; the initial glucose concentration is high, and the biomass is too high after the base sugar is consumed, then the bacteria are in a state of aging, the vitality decreases, the induction effect decreases, and the ectoine content is also low. This is called the Klebsiella pneumoniae effect.

[0140] The Klebsiella effect, also known as the glucose effect, refers to the fact that the metabolites of glucose will reduce the cAMP level. On the one hand, the CAP protein of cAMP binds to the CAP binding site on DNA, so that RNA polymerase can bind to the promoter region of the lactose operon and start transcription; on the other hand, cAMP acts as a second messenger to phosphorylate target cell proteins by activating APK (cAMP-dependent protein kinase), thereby regulating cell responses. Therefore, when the glucose content in the fermentation broth is high, even if there is an inducer, it will have an adverse effect on the induction and is not conducive to the expression of the enzyme. Therefore, generally, fermentation requires induction after the substrate sugar is consumed, and the substrate sugar concentration is crucial to the synthesis of the enzyme that needs to be induced.

[0141] Experimental Example 4

[0142] Recombinant strain BL21(DE3) / (pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd) fermentation process, after the substrate sugar was consumed, different OD growth rates were regulated, and other process conditions were the same as Example 8. The fermentation content of ectoine was determined, and the results are listed in Table 5.

[0143] Table 5 Effect of different OD growth rates

[0144]

[0145] As shown in Table 5, when the OD growth rate is 0-2 / h, the ectoine fermentation content is 50.5g / L, when the OD growth rate is 2-4 / h, the ectoine fermentation content is 75.6g / L, when the OD growth rate is 4-6 / h, the ectoine fermentation content is 63.4g / L, and when the OD growth rate is 7-8 / h, the ectoine fermentation content is 35.8g / L. It can be seen that when the OD growth rate is 2-4 / h, the ectoine fermentation content is the highest. This is because after the addition of the inducer, the strain begins to transform from reproductive growth to the synthesis of ectoine-related enzymes. The OD growth rate is controlled by adding glucose. If the OD growth is too slow, the inducer will cause greater damage to the cells, resulting in a low final biomass and a low ectoine content; if the OD growth is too fast, the induction effect will be poor, the expression of ectoine-related enzymes will be low, and the ectoine fermentation content will be low. After the base sugar is consumed, the addition of glucose solution to control the OD growth rate to 2-4 can balance the reproductive growth of the strain and the expression of ectoine-related enzymes, and the ectoine fermentation content is the highest.

[0146] Experimental Example 5

[0147] Recombinant strain BL21(DE3) / (pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd) fermentation process, when OD grew to 45-65, the sugar supplement rate was accelerated to adjust the sugar concentration to 0.2-1g / L, and other process conditions were the same as those in Example 8. The fermentation content of ectoine was determined, and the results are listed in Table 6.

[0148] Table 6 Effect of increasing sugar supplement rate at different OD

[0149]

[0150] Due to the glucose effect, when the glucose content in the fermentation broth is high, even the presence of inducer will have an adverse effect on the induction, so the induced expression of ectoine synthesis-related enzymes is carried out at extremely low glucose concentrations. However, the substrate for ectoine synthesis is glucose, so the expression of ectoine-related enzymes and the synthesis of ectoine need to be regulated by adjusting the glucose concentration in the fermentation broth.

[0151] As shown in Table 6, when the sugar addition rate was accelerated at OD of 45, the fermentation content of ectoine was 62.4 g / L, when the sugar addition rate was accelerated at OD of 55, the fermentation content of ectoine was 75.6 g / L, and when the sugar addition rate was accelerated at OD of 65, the fermentation content of ectoine was 53.7 g / L. It can be seen that when a glucose solution with a concentration of 700 g / L was added to the fermentation OD of 55, the sugar concentration was adjusted to 0.2-1 g / L, and the fermentation content of ectoine was the highest.

[0152] Experimental Example 6

[0153] Recombinant strain BL21(DE3) / (pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd) fermentation process, different initial pH was adjusted, other process conditions were the same as Example 8, and the fermentation content of ectoine was measured. The measurement results are listed in Table 7.

[0154] Table 7 Effect of different initial pH

[0155]

[0156] When the substrate sugar is consumed, the strain grows actively. The OD growth rate is regulated to 2-4 / h by adding a glucose solution with a concentration of 700g / L. The insufficient sugar supply will cause the fermentation pH to rise slowly by 0.3-0.5. Too high a pH will cause the strain activity and enzyme activity to decrease, affecting the fermentation content of ectoine. As shown in Table 7, when the initial pH is 6.8, the fermentation content of ectoine is 70.4g / L, when the initial pH is 7.0, the fermentation content of ectoine is 75.6g / L, when the initial pH is 7.2, the fermentation content of ectoine is 63.9g / L, and when the initial pH is 7.4, the fermentation content of ectoine is 51.5g / L. When the initial pH is 7.0, the fermentation content of ectoine is the highest.

[0157] Experimental Example 7

[0158] Recombinant strain BL21(DE3) / (pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd) fermentation process, the pH after different induction was adjusted, and other process conditions were the same as Example 8. The fermentation content of ectoine was determined, and the results are listed in Table 8.

[0159] Table 8 Effect of pH after different induction

[0160]

[0161] The optimal pH for Escherichia coli culture is neutral or neutral to alkaline, but the enzyme activity is different under different pH conditions. It can be seen from the data in Table 8 that when the initial pH is 7.0, the fermentation content of ectoine is 58.7 g / L when the pH is 6.8 after induction, the fermentation content of ectoine is 63.5 g / L when the pH is 7.0 after induction, the fermentation content of ectoine is 70.8 g / L when the pH is 7.2 after induction, and the fermentation content of ectoine is 75.6 g / L when the pH is 7.4 after induction. It can be seen that when the pH is 7.4 after induction, the enzyme activity is high and the fermentation content of ectoine is the highest.

[0162] In summary, the recombinant strain BL21(DE3) / (pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd) fermentation to produce ectoine, by optimizing the substrate sugar concentration, OD growth rate, accelerating the sugar replenishment time and fermentation pH, and controlling the fermentation process, the synthesis efficiency of ectoine is improved, the fermentation content of ectoine is high, and the production cost of ectoine is low, which is conducive to the promotion and application of ectoine.

[0163] It can be seen that the present invention uses the ectoine ectoine gene cluster and mutants to construct a recombinant strain, so that the synthesis efficiency and content of ectoine produced by fermentation are improved, which is beneficial to increasing the yield of ectoine, reducing the production cost of ectoine, and facilitating the promotion and utilization of ectoine.

Claims

1. A quaternary pyrimidine gene cluster mutant derived from halophilic Bacillus, characterized in that: The nucleic acid sequence of the mutant is shown in SEQ ID NO.

8.

2. The use of the mutant according to claim 1 in the process of synthesizing ectoine, characterized in that: The following steps are involved: (1) constructing a recombinant vector containing the ectoine gene cluster mutant according to claim 1; (2) constructing a recombinant strain containing the recombinant vector of step (1); (3) Fermentation to produce tetrahydropyrimidine using glucose as substrate.

3. The use according to claim 2, characterized in that: Step (3) includes seed plate activation, seed liquid preparation, and fermentation to produce ectoine, wherein the specific operation of fermentation to produce ectoine is as follows: the seed liquid is connected to a fermentation tank containing 8L of fermentation medium, the temperature is 37°C, the dissolved oxygen is 20-30%, the initial pH is regulated to 7.0 with ammonia water, after the substrate sugar is consumed, IPTG is added to the fermentation medium to a concentration of 0.1mM / L, a glucose solution with a concentration of 700g / L is added, the growth rate of OD is regulated to 2-6 / h, ammonia water is added to regulate the pH to 7.4, and when the OD grows to 55, the sugar addition rate is accelerated to a sugar concentration of 0.2-1g / L, and the fermentation is terminated after 30h to obtain a fermentation liquid containing ectoine.

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