CN118006645A - Tetrahydropyrimidine gene cluster derived from bacillus salicillus, mutant and application - Google Patents
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Abstract
The invention discloses a tetrahydropyrimidine gene cluster derived from bacillus salicillus, a mutant and application thereof, and belongs to the technical field of synthetic biology. The tetrahydropyrimidine gene cluster nucleic acid sequence is shown as SEQ ID NO.7, and comprises the following 3 genes: ectA gene, ectB gene, ectC gene, and nucleotide sequence shown in SEQ ID NO.2, 3, and optimizing the design mutation of the gene cluster to obtain mutant, wherein the nucleotide sequence is shown in SEQ ID NO. 8. The recombinant strain constructed by the tetrahydropyrimidine gene cluster and the mutant thereof can improve the synthesis efficiency of the tetrahydropyrimidine, improve the fermentation content of the tetrahydropyrimidine, reduce the production cost of the tetrahydropyrimidine and be beneficial to popularization and application of the tetrahydropyrimidine.
Description
Technical Field
The invention relates to a tetrahydropyrimidine gene cluster derived from bacillus salicillus, a mutant and application thereof, belonging to the technical field of synthetic biology.
Background
Tetrahydropyrimidine (1, 4,5, 6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid, C 6H10N2O2,MW = 142.16) is a cyclic amino acid derivative, which is discovered for the first time from photosynthetic rhodobacter sphaeroides Halochloris of the genus extreme halophiles rhodospirillum (Ectothiorhodspira), is discovered to be ubiquitous in halophilic or salt-tolerant microorganisms, has strong water molecule complexing capability, has good protective effects on proteins, nucleic acids, enzymes, biological membranes and cells under adverse conditions such as high osmotic pressure, high temperature, low temperature, drying, strong acid and alkali, radiation and the like, and has wide application prospect in the fields of medicines, cosmetics, biological preparations and the like at present.
The synthesis of the tetrahydropyrimidine mainly comprises a chemical method and a biological method, and is widely adopted due to the characteristics of simple production process, high synthesis rate, low separation and purification difficulty in the later stage, environmental friendliness and the like of the biological synthesis method compared with the chemical synthesis method. The biosynthesis components of tetrahydropyrimidine are three steps: firstly, L-aspartic acid-beta-semialdehyde (L-aspartate-beta-SEMIALDEHYDE, ASA) is taken as a substrate, and is catalyzed by 2, 4-diaminobutyric acid aminotransferase (diaminobutyric acid aminoransferase, ectB) to generate L-2, 4-diaminobutyric acid (L-2, 4-diaminobutyrate, DABA); secondly, under the catalysis of 2, 4-diaminobutyric acid acetyl transferase (diaminobutyric ACID ACETYLTRANSFERASE, ectA), DABA is acetylated to generate N-acetyl-L-2, 4-diaminobutyric acid (N-gamma-acetyl-L-2, 4-diaminobutyrate, ADABA); finally, ADABA was cyclized by an ectoine synthase (EctC) to form tetrahydropyrimidine.
The early stage of the biosynthesis of the tetrahydropyrimidine is produced by a halophagomonas 'bacterial milking' mode, namely, firstly, culturing cells under high osmotic pressure and accumulating the tetrahydropyrimidine in the cells, then, stimulating the release of the tetrahydropyrimidine from the cells to the outside through hypotonic impact, and then, repeatedly performing hypertonic culture and hypotonic impact to obtain the high-concentration tetrahydropyrimidine. This process is complicated to operate and requires cultivation under high salt conditions, and thus requires high equipment requirements, and high salt waste liquid discharge causes environmental problems, resulting in very expensive production.
In recent years, researchers construct engineering strains by heterologously expressing gene clusters of tetrahydropyrimidine in escherichia coli and corynebacterium glutamicum, so that the fermentation difficulty is reduced, and the fermentation content of the tetrahydropyrimidine is improved. However, the tetrahydropyrimidine gene cluster used for constructing engineering strains is mostly derived from halophagomonas such as: h. elongate DSM 2581, gene cluster sequences are highly conserved. Therefore, the development of new tetrahydropyrimidine gene resources improves the fermentation content of the tetrahydropyrimidine, reduces the production cost and improves the production efficiency, and has important practical significance for the application of the tetrahydropyrimidine.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a tetrahydropyrimidine gene cluster, a mutant and application of the gene cluster and the mutant which are derived from bacillus salicifolius, wherein the gene cluster and the mutant are used for constructing a recombinant tetrahydropyrimidine engineering strain, and the content of the tetrahydropyrimidine obtained by fermentation is high.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
The invention provides a tetrahydropyrimidine gene cluster derived from bacillus salicillus, the nucleic acid sequence of the tetrahydropyrimidine gene cluster is shown as SEQ ID NO.7, and the tetrahydropyrimidine gene cluster comprises the following 3 genes: ectA gene, the nucleic acid sequence is shown as SEQ ID NO. 1; ectB gene, the nucleic acid sequence is shown as SEQ ID NO. 2; ectC gene and nucleic acid sequence shown in SEQ ID NO. 3. The ectA, ectB, ectC gene sequences of the tetrahydropyrimidine gene cluster are different from the gene sequences in the database, so that the novel tetrahydropyrimidine synthetic gene sequence is a novel tetrahydropyrimidine synthetic gene sequence.
Further, the tetrahydropyrimidine gene cluster is derived from bacillus caldus Halobacillus sp, FL-2423 with tetrahydropyrimidine secretion characteristics, and the strain is preserved in China general microbiological culture collection center (CGMCC) No.18783 in 11/01/2019, and the preservation address is China academy of microorganisms (national academy of sciences) No. 3 of the West way 1 of the Chat area North Star of Beijing city.
The invention also provides a mutant formed by the tetrahydropyrimidine gene cluster derived from the bacillus halophilus, and the nucleic acid sequence of the mutant is shown as SEQ ID NO. 8.
Further, the mutant consists of amino acid sequences shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.
Furthermore, the amino acid sequence shown in SEQ ID NO.4 is a ectA gene original sequence shown in SEQ ID NO.1, and has the following mutation: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is replaced by I from L at the 54 th amino acid and by T from Q at the 103 th amino acid.
Furthermore, the amino acid sequence shown in SEQ ID NO.5 is a ectB gene original sequence with a nucleic acid sequence shown in SEQ ID NO.2, and has the following mutation: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is replaced by Q at the 28 th amino acid, P at the 67 th amino acid, T at the 189 th amino acid, H at the 251 nd amino acid, and E at the 331 st amino acid.
Furthermore, the amino acid sequence shown in SEQ ID NO.6 is a ectC gene original sequence shown in SEQ ID NO.3, and has the following mutation on the basis of the original sequence: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is replaced by E from A at the 46 th amino acid, R from D at the 90 th amino acid and E from Q at the 134 th amino acid.
The invention also provides an application of the bacillus halophilus-derived tetrahydropyrimidine gene cluster and a mutant formed by the same in the process of synthesizing the tetrahydropyrimidine, which comprises the following steps:
(1) Obtaining a bacillus salicillus FL-2423 tetrahydropyrimidine gene cluster;
(2) Preparing bacillus salicillus FL-2423 tetrahydropyrimidine gene cluster mutants;
(3) Constructing a recombinant vector containing FL-2423 tetrahydropyrimidine gene cluster or a tetrahydropyrimidine gene cluster mutant;
(4) Constructing a tetrahydropyrimidine recombinant strain;
(5) And fermenting with glucose as a substrate to produce the tetrahydropyrimidine.
Note that: IPTG is isopropyl-beta-D-thiogalactoside.
Further, the specific operation of step (1) is as follows:
(1-1) inoculating the activated FL-2423 strain into a high-salt culture medium, culturing for 16 hours at 28 ℃, extracting genome DNA by using a bacterial genome extraction kit, and sequencing the genome of the organisms of the family Prinsepia;
(1-2) searching nucleic acid and amino acid sequences of Halomonas elongata DSM and 2581 tetrahydropyrimidine genes ectA (GenBank record: CBV 42472.1), ectB (GenBank record: CBV 42473.1) and ectC (GenBank record: CBV 42474.1) from NCBI database;
(1-3) performing homology comparison analysis on ectA, ectB, ectC nucleic acid and amino acid sequences of DSM 2581 and genome sequencing results and gene structure annotation results of FL-2423 respectively to obtain ectA, ectB, ectC nucleic acid sequence of FL-2423;
(1-4) subjecting the ectA, ectB, ectC nucleic acid sequence of FL-2423 to tree analysis using NCBI Nucleotide BLAST in NCBI database, respectively, and the results are shown in FIGS. 1,2 and 3;
Further, in the step (1-1), the high-salt culture medium comprises 30.0g/L of sodium chloride, 10.0g/L of glucose, 1.0g/L of yeast powder, 2.5g/L of peptone, 2.0g/L of disodium hydrogen phosphate, 2.0g/L of sodium dihydrogen phosphate, 0.85g/L of potassium chloride, 0.4g/L of magnesium sulfate, 0.01g/L of manganese sulfate, 0.01g/L of ferrous sulfate, 20.0g/L of calcium carbonate and pH of 7.0.
Further, the specific operation of the step (2) is as follows: the genome of FL-2423 is used as a template, a primer 2423ectABC-F/ectABC-R is used for amplifying a tetrahydropyrimidine gene cluster to obtain a FL-2423-ectABC fragment, a site-directed mutagenesis design is carried out on the FL-2423-ectABC, and a mutated FL-2423-ectABC mut fragment is obtained by gene synthesis.
Further, the specific operation of the step (3) is as follows: amplifying the obtained linearization fragments by using a primer by using a plasmid as a template; the FL-2423-ectABC or FL-2423-ectABC mut is used as a template, the FL-2423-ectABC or FL-2423-ectABC mut gene cluster containing a carrier homology arm is obtained by amplification, the linearization fragment and the tetrahydropyrimidine gene cluster are connected by homologous recombinant enzyme, the linearization fragment and the tetrahydropyrimidine gene cluster are transformed into escherichia coli DH5 alpha, and after resistance screening, plasmids are extracted, so that the recombinant expression carrier is obtained.
Further, in step (3), the resistance screen is an ampicillin resistance screen or a kana resistance screen.
Further, the specific operation of step (4) is as follows: the expression vector is transformed into competent cells of escherichia coli BL21 (DE 3) by a thermal shock method to obtain a recombinant strain. Wherein the temperature of the thermal shock method is 42 ℃ and the time is 90s.
Further, the step (5) and the step (5) comprise seed plate activation, seed liquid preparation and fermentation production of tetrahydropyrimidine, and the specific operations are as follows:
(5-1) plate seed activation: scribing the recombinant strain of the tetrahydropyrimidine on an eggplant-shaped bottle containing a resistant solid LB culture medium in an ultra-clean workbench, and culturing at a constant temperature of 37 ℃ for 12 hours to obtain activated seeds;
(5-2) seed liquid preparation: inoculating activated seeds in the eggplant-shaped bottle into a fermentation tank filled with 10L of seed culture medium, regulating pH to 7.0 with ammonia water at 37 ℃ and dissolved oxygen of 20-30%, and culturing until OD is 15 to obtain seed liquid;
(5-3) fermenting to produce tetrahydropyrimidine: the seed liquid is put into a fermentation tank filled with 8L of fermentation medium, the temperature is 37 ℃, dissolved oxygen is 20-30%, ammonia water is used for regulating and controlling the initial pH value to 7.0, IPTG is added to the fermentation medium until the concentration is 0.1mM/L after the bottom sugar is consumed, glucose solution with the concentration of 700g/L is fed, the growth rate of OD is regulated and controlled to be 2-6/h, ammonia water is added for regulating and controlling the pH value to 7.4, the sugar supplementing speed is accelerated until the sugar concentration is 0.2-1g/L when the OD grows to 55, and fermentation is finished for 30h, so that the fermentation liquid containing tetrahydropyrimidine is obtained.
Further, in the step (5-2), the seed culture medium comprises the following components: 30.0g/L glucose, 1.5g/L peptone, 6.8g/L yeast powder, 0.68g/L citric acid monohydrate, 1.6g/L potassium dihydrogen phosphate, 0.68g/L magnesium sulfate heptahydrate and 0.33g/L GPE defoamer.
Further, in the step (5-3), the seed liquid accounts for 15% of the fermentation medium by volume.
Further, in the step (5-3), the components of the fermentation medium are: 25.0g/L of glucose, 2.5g/L of peptone, 3.8g/L of yeast powder, 6.8g/L of corn steep liquor dry powder, 2.5g/L of citric acid monohydrate, 3.8g/L of potassium dihydrogen phosphate, 1.5g/L of magnesium sulfate heptahydrate and 0.85g/L of GPE defoamer. When the glucose content in the fermentation liquid is high, even if an inducer exists, the inducer can have adverse effect on induction and is unfavorable for enzyme expression, so that the induction is generally needed after the consumption of the bottom sugar in the fermentation, and the concentration of the bottom sugar is important for the synthesis of the enzyme which needs to be induced for expression. The addition of the inducer can inhibit the growth of cells, the initial glucose concentration is low, the OD is small during induction, the biomass is low during induction, the thalli cannot grow to a higher cell concentration, and the tetrahydropyrimidine content is low; the initial glucose concentration is high, biomass is too high after the bottom sugar is consumed, so that the thalli are in an aging state, activity is reduced, induction effect is reduced, and the content of the tetrahydropyrimidine is low.
Further, in the step (5-3), IPTG is added to the fermentation medium until the concentration is 0.1mM/L after the bottom sugar is consumed, a glucose solution with the concentration of 700g/L is fed, and the OD growth rate is regulated to be 2-4/h. After addition of the inducer, the strain was transformed from reproductive growth to the enzyme involved in the synthesis of tetrahydropyrimidine, and the OD growth rate was controlled by fed-batch glucose. When OD grows too slowly, the inducer is more harmful to cells, resulting in lower final biomass and lower tetrahydropyrimidine content; when OD grows too fast, the induction effect is poor, the expression quantity of the tetrahydropyrimidine related enzyme is low, and the fermentation content of the tetrahydropyrimidine is low.
Compared with the prior art, the invention has the beneficial effects that:
1. The tetrahydropyrimidine gene cluster provided by the invention is derived from bacillus salicifolius Halobacillus sp, FL-2423 with tetrahydropyrimidine secretion characteristics, and is a novel tetrahydropyrimidine synthetic gene sequence after NCBI Nucleotide BLAST analysis and is different from the existing encoding sequence of the tetrahydropyrimidine gene.
2. The coded enzyme catalysis production efficiency of the tetrahydropyrimidine gene cluster of FL-2423 provided by the invention is better than that of a commonly applied tetrahydropyrimidine gene cluster of Halomonas elongata DSM and 2581, and the tetrahydropyrimidine content is improved by more than 20% under the same fermentation condition.
3. The invention carries out mutation design on the tetrahydropyrimidine gene cluster from the bacillus salicillus to obtain a mutant formed by the tetrahydropyrimidine gene cluster, the activity is enhanced, the synthesis efficiency of the tetrahydropyrimidine is greatly improved, and under the same fermentation condition, compared with a strain constructed by Halobacillus sp, FL-2423 original tetrahydropyrimidine gene cluster, the fermentation tetrahydropyrimidine content of the mutant formed by the tetrahydropyrimidine gene cluster is improved by more than 30 percent; compared with the strain constructed by Halomonas elongata DSM and 2581 tetrahydropyrimidine gene cluster, the tetrahydropyrimidine fermentation content of the mutant recombinant strain is improved by more than 60%.
4. The application of the tetrahydropyrimidine gene cluster and the mutant from the bacillus salicils improves the synthesis efficiency of the tetrahydropyrimidine, thereby improving the fermentation content of the tetrahydropyrimidine, reducing the production cost of the tetrahydropyrimidine and being beneficial to popularization and application of the tetrahydropyrimidine.
Drawings
FIG. 1 is a FL-2423 ectA gene phylogenetic tree analysis;
FIG. 2 is a FL-2423 ectB gene phylogenetic tree analysis;
FIG. 3 is a FL-2423 ectC gene phylogenetic tree analysis.
Detailed Description
The principles and features of the present invention are described below with examples given for the purpose of illustration only and are not intended to limit the scope of the invention. Meanwhile, the experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents, materials, and apparatus, unless otherwise specified, are all of the prior art and are commercially available.
EXAMPLE 1 acquisition of Halobacillus sp.FL-2423 tetrahydropyrimidine Gene Cluster
(1) Inoculating the activated FL-2423 strain into a high-salt culture medium, culturing for 16 hours at 28 ℃, extracting genome DNA by using a bacterial genome extraction kit, sequencing a genome of a plant of the family Praeparataceae, and annotating a gene structure, wherein the high-salt culture medium comprises the following components: 30.0g/L of sodium chloride, 10.0g/L of glucose, 1.0g/L of yeast powder, 2.5g/L of peptone, 2.0g/L of disodium hydrogen phosphate, 2.0g/L of sodium dihydrogen phosphate, 0.85g/L of potassium chloride, 0.4g/L of magnesium sulfate, 0.01g/L of manganese sulfate, 0.01g/L of ferrous sulfate, 20.0g/L of calcium carbonate and pH of 7.0;
(2) The nucleotide and amino acid sequences of Halomonas elongata DSM and 2581 tetrahydropyrimidine genes ectA (GenBank record: CBV 42472.1), ectB (GenBank record: CBV 42473.1) and ectC (GenBank record: CBV 42474.1) are searched from NCBI database;
(3) Carrying out homology comparison analysis on the ectA, ectB, ectC nucleic acid sequence and the amino acid sequence of DSM 2581 and the genome sequencing result and the gene structure annotation result of FL-2423 respectively to obtain a ectA, ectB, ectC nucleic acid sequence of FL-2423, wherein the nucleic acid sequence is shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3;
(4) The nucleic acid sequence ectA, ectB, ectC of FL-2423 was analyzed by the row tree using NCBI Nucleotide BLAST in NCBI database, and the results are shown in FIGS. 1,2 and 3, respectively.
SEQ ID NO.1：FL-2423-ectA
atgaacgtgaccacagagcccttcattccctccgccgagctcgcccggcccagcattgccgatgccgtggtcggccatcaggccacgccgctgttcatccgcaagcccagccccgacgacggctggggcatctacgaactggtcaagtcctgcccgccgctggacgtcaattccgcctatgcctacctgctgctggccacccagttccgcgacagctgtgccgtggctaccaacgaggagggcgagatcgtcggcttcgtttccggctacgtgaagagcaactgcccggacacctatttcctctggcaggtcgcggtgggtgagaaggcgcgcggcaccggcctggcccgccgcctggtcgaagccgtgatgacccgcccggagatggccgaggtgcaccacctcgagaccaccatcactcccgacaaccaggcatcctgggggctcttccggcgcctcgccgaccgctggcaggcgccgctcaacagccgcgagtacttctccaccgaccagctcggcggcgagcatgacccggaaaacctcgtccgcatcggtcccttccagaccgaccagatctga
SEQ ID NO.2：FL-2423-ectB
atgcagacccagattctcgaacgcatggagtccgaggttcggacctattcacgctcctttccggtggtcttcaccaaggcccggaatgcccgtctgaccgacgaggacggccgcgagtacatcgatttcctggccggcgccggcaccctgaactacggccacaacaaccccaagatcaagcaggcgctggtcgactacctggcctccgacggcatcgtccacgggctggacttctggaccgcggccaagcgtgactacctcgagaccctggaggaggtgatcctcaagccgcggggcctggactacaaggtgcatctgccggggccgaccggtaccaacgccgtcgaagcggcgatccgtctggcgcgggtggccaaagggcgccacaacatcgtcaccttcaccaacggctttcacggtgtcaccatgggcgcgctggcgaccaccggtaaccgcaagttccgcgaggccaccggtggcatcccgacccagggcgcaagcttcttgccttatgacggctacatgggcgagcacaccgataccctcgactacttcgagaagctgctgggtgacaaatccggcggcctcgacctgcccgcggcggtgatcgtcgagaccgttcagggcgagggcggcatcaatgtggctggtctggattggctcaagcgcctggaaggcatctgccgggcccatgacatcctgttgatcgtcgatgacatcccagccggctgcggccgcaccggcaagttcttcagcttcgagcacgccggcgtgacccgcgatattgtgaccaactccaagtctctgtccggttacggcctgccgttcgctcacgtcctgatgcgccccgagctcgacaagtggaagcccggccagtacaacggcaccttccgcggcttcaacctggccttcaccaccgccgccgcgacgctgcgccagtactggagcgatgacgtcttcgagcgcgatgtccagcgcaagggccgcgtggtcgccgaccgcttccagaagatcgccgcctggctgagcgagaacggcatcgaggcctccgagcgtggccgcgggctgatgcgtggcatcgacgtgggttccggcgatattgccgacaagatcacccaccaagccttcgagaacgggttggtcatcgagaccagcggccaggacggtgaggtggtcaagtgcctgtgcccgctgaccatccccgacgaggacctgatcgaggccctggacattctcgaggccagcgcccgccaggccctgagctga
SEQ ID NO.3：FL-2423-ectC
atgatcgttcgcaacctcgaagtcgcccgcgagaccgaccgtctggtcaccgccgaaaacggcaactgggacagcacccgcctgtctctggccgaagatggtggcaactgctccttccacatcacccgtatctatgccggcaccgagacccatatccactacaagcatcacttcgaggcggtttattgcatcgaaggcgagggcgaagtggaaaccctggccgatggcaagatctggccgatcaagccgggcgacatctatatcctcgaccagcacgacgagcacctgctgcgggcgtacaagaccatgcacctggcctgcgtgttcacgccgggcctgaccggcaacgaggtgcaccgcgaagacggttcctacgcacctgccgacgaagccgacgaccagaagccgctgtaa
EXAMPLE 2 preparation of Halobacillus sp, FL-2423 tetrahydropyrimidine Gene cluster mutants
The genome of FL-2423 is used as a template, a primer 2423ectABC-F/ectABC-R is used for amplifying a tetrahydropyrimidine gene cluster to obtain a FL-2423-ectABC fragment, a nucleic acid sequence is shown as SEQ ID NO.7, a site-directed mutagenesis design is carried out on ectABC, the FL-2423-ectABC mut fragment after gene synthesis mutation is shown as SEQ ID NO.8, and an amino acid sequence is shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.
SEQ ID NO.4
MNVTTEPFIPSAELARPSIADAVVGHQATPLFIRKPSPDDGWGIYELVKSCPPIDVNSAYAYLLLATQFRDSCAVATNEEGEIVGFVSGYVKSNCPDTYFLWTVAVGEKARGTGLARRLVEAVMTRPEMAEVHHLETTITPDNQASWGLFRRLADRWQAPLNSREYFSTDQLGGEHDPENLVRIGPFQTDQI*
SEQ ID NO.5
MQTQILERMESEVRTYSRSFPVVFTKAQNARLTDEDGREYIDFLAGAGTLNYGHNNPKIKQALVDYPASDGIVHGLDFWTAAKRDYLETLEEVILKPRGLDYKVHLPGPTGTNAVEAAIRLARVAKGRHNIVTFTNGFHGVTMGALATTGNRKFREATGGIPTQGASFLPYDGYMGEHTDTLDYFEKLTGDKSGGLDLPAAVIVETVQGEGGINVAGLDWLKRLEGICRAHDILLIVDDIPAGCGRTGKFHSFEHAGVTRDIVTNSKSLSGYGLPFAHVLMRPELDKWKPGQYNGTFRGFNLAFTTAAATLRQYWSDDVFERDVQRKGRVEADRFQKIAAWLSENGIEASERGRGLMRGIDVGSGDIADKITHQAFENGLVIETSGQDGEVVKCLCPLTIPDEDLIEALDILEASARQALS*
SEQ ID NO.6
MIVRNLEVARETDRLVTAENGNWDSTRLSLAEDGGNCSFHITRIYEGTETHIHYKHHFEAVYCIEGEGEVETLADGKIWPIKPGDIYILRQHDEHLLRAYKTMHLACVFTPGLTGNEVHREDGSYAPADEADDEKPL*
SEQ ID NO.7: halobacillus sp, FL-2423 tetrahydropyrimidine gene cluster
atgaacgtgaccacagagcccttcattccctccgccgagctcgcccggcccagcattgccgatgccgtggtcggccatcaggccacgccgctgttcatccgcaagcccagccccgacgacggctggggcatctacgaactggtcaagtcctgcccgccgctggacgtcaattccgcctatgcctacctgctgctggccacccagttccgcgacagctgtgccgtggctaccaacgaggagggcgagatcgtcggcttcgtttccggctacgtgaagagcaactgcccggacacctatttcctctggcaggtcgcggtgggtgagaaggcgcgcggcaccggcctggcccgccgcctggtcgaagccgtgatgacccgcccggagatggccgaggtgcaccacctcgagaccaccatcactcccgacaaccaggcatcctgggggctcttccggcgcctcgccgaccgctggcaggcgccgctcaacagccgcgagtacttctccaccgaccagctcggcggcgagcatgacccggaaaacctcgtccgcatcggtcccttccagaccgaccagatctgagccgggacgccgcctggccggcccggtacgggggcagagcacccctttatccgccgatttttcccgacaggaggtcgcaaaacaggaggtcgctcatgcagacccagattctcgaacgcatggagtccgaggttcggacctattcacgctcctttccggtggtcttcaccaaggcccggaatgcccgtctgaccgacgaggacggccgcgagtacatcgatttcctggccggcgccggcaccctgaactacggccacaacaaccccaagatcaagcaggcgctggtcgactacctggcctccgacggcatcgtccacgggctggacttctggaccgcggccaagcgtgactacctcgagaccctggaggaggtgatcctcaagccgcggggcctggactacaaggtgcatctgccggggccgaccggtaccaacgccgtcgaagcggcgatccgtctggcgcgggtggccaaagggcgccacaacatcgtcaccttcaccaacggctttcacggtgtcaccatgggcgcgctggcgaccaccggtaaccgcaagttccgcgaggccaccggtggcatcccgacccagggcgcaagcttcttgccttatgacggctacatgggcgagcacaccgataccctcgactacttcgagaagctgctgggtgacaaatccggcggcctcgacctgcccgcggcggtgatcgtcgagaccgttcagggcgagggcggcatcaatgtggctggtctggattggctcaagcgcctggaaggcatctgccgggcccatgacatcctgttgatcgtcgatgacatcccagccggctgcggccgcaccggcaagttcttcagcttcgagcacgccggcgtgacccgcgatattgtgaccaactccaagtctctgtccggttacggcctgccgttcgctcacgtcctgatgcgccccgagctcgacaagtggaagcccggccagtacaacggcaccttccgcggcttcaacctggccttcaccaccgccgccgcgacgctgcgccagtactggagcgatgacgtcttcgagcgcgatgtccagcgcaagggccgcgtggtcgccgaccgcttccagaagatcgccgcctggctgagcgagaacggcatcgaggcctccgagcgtggccgcgggctgatgcgtggcatcgacgtgggttccggcgatattgccgacaagatcacccaccaagccttcgagaacgggttggtcatcgagaccagcggccaggacggtgaggtggtcaagtgcctgtgcccgctgaccatccccgacgaggacctgatcgaggccctggacattctcgaggccagcgcccgccaggccctgagctgaacggcaaggccggccatccgccgcacggggtgtatcggtcctcggccggtcgcggcctcgccttggccgacccgtcatagacactactggagagccaccatgatcgttcgcaacctcgaagtcgcccgcgagaccgaccgtctggtcaccgccgaaaacggcaactgggacagcacccgcctgtctctggccgaagatggtggcaactgctccttccacatcacccgtatctatgccggcaccgagacccatatccactacaagcatcacttcgaggcggtttattgcatcgaaggcgagggcgaagtggaaaccctggccgatggcaagatctggccgatcaagccgggcgacatctatatcctcgaccagcacgacgagcacctgctgcgggcgtacaagaccatgcacctggcctgcgtgttcacgccgggcctgaccggcaacgaggtgcaccgcgaagacggttcctacgcacctgccgacgaagccgacgaccagaagccgctgtaa
SEQ ID NO.8: halobacillus sp, FL-2423 tetrahydropyrimidine gene cluster mutants
atgaacgtgaccacagagcccttcattccctccgccgagctcgcccggcccagcattgccgatgccgtggtcggccatcaggccacgccgctgttcatccgcaagcccagccccgacgacggctggggcatctacgaactggtcaagtcctgcccgccgatcgacgtcaattccgcctatgcctacctgctgctggccacccagttccgcgacagctgtgccgtggctaccaacgaggagggcgagatcgtcggcttcgtttccggctacgtgaagagcaactgcccggacacctatttcctctggacggtcgcggtgggtgagaaggcgcgcggcaccggcctggcccgccgcctggtcgaagccgtgatgacccgcccggagatggccgaggtgcaccacctcgagaccaccatcactcccgacaaccaggcatcctgggggctcttccggcgcctcgccgaccgctggcaggcgccgctcaacagccgcgagtacttctccaccgaccagctcggcggcgagcatgacccggaaaacctcgtccgcatcggtcccttccagaccgaccagatctgagccgggacgccgcctggccggcccggtacgggggcagagcacccctttatccgccgatttttcccgacaggaggtcgcaaaacaggaggtcgctcatgcagacccagattctcgaacgcatggagtccgaggttcggacctattcacgctcctttccggtggtcttcaccaaggcccagaatgcccgtctgaccgacgaggacggccgcgagtacatcgatttcctggccggcgccggcaccctgaactacggccacaacaaccccaagatcaagcaggcgctggtcgactacccggcctccgacggcatcgtccacgggctggacttctggaccgcggccaagcgtgactacctcgagaccctggaggaggtgatcctcaagccgcggggcctggactacaaggtgcatctgccggggccgaccggtaccaacgccgtcgaagcggcgatccgtctggcgcgggtggccaaagggcgccacaacatcgtcaccttcaccaacggctttcacggtgtcaccatgggcgcgctggcgaccaccggtaaccgcaagttccgcgaggccaccggtggcatcccgacccagggcgcaagcttcttgccttatgacggctacatgggcgagcacaccgataccctcgactacttcgagaagctgacgggtgacaaatccggcggcctcgacctgcccgcggcggtgatcgtcgagaccgttcagggcgagggcggcatcaatgtggctggtctggattggctcaagcgcctggaaggcatctgccgggcccatgacatcctgttgatcgtcgatgacatcccagccggctgcggccgcaccggcaagttccacagcttcgagcacgccggcgtgacccgcgatattgtgaccaactccaagtctctgtccggttacggcctgccgttcgctcacgtcctgatgcgccccgagctcgacaagtggaagcccggccagtacaacggcaccttccgcggcttcaacctggccttcaccaccgccgccgcgacgctgcgccagtactggagcgatgacgtcttcgagcgcgatgtccagcgcaagggccgcgtggaggccgaccgcttccagaagatcgccgcctggctgagcgagaacggcatcgaggcctccgagcgtggccgcgggctgatgcgtggcatcgacgtgggttccggcgatattgccgacaagatcacccaccaagccttcgagaacgggttggtcatcgagaccagcggccaggacggtgaggtggtcaagtgcctgtgcccgctgaccatccccgacgaggacctgatcgaggccctggacattctcgaggccagcgcccgccaggccctgagctgaacggcaaggccggccatccgccgcacggggtgtatcggtcctcggccggtcgcggcctcgccttggccgacccgtcatagacactactggagagccaccatgatcgttcgcaacctcgaagtcgcccgcgagaccgaccgtctggtcaccgccgaaaacggcaactgggacagcacccgcctgtctctggccgaagatggtggcaactgctccttccacatcacccgtatctatgagggcaccgagacccatatccactacaagcatcacttcgaggcggtttattgcatcgaaggcgagggcgaagtggaaaccctggccgatggcaagatctggccgatcaagccgggcgacatctatatcctccgccagcacgacgagcacctgctgcgggcgtacaagaccatgcacctggcctgcgtgttcacgccgggcctgaccggcaacgaggtgcaccgcgaagacggttcctacgcacctgccgacgaagccgacgacgagaagccgctgtaa
EXAMPLE 3 construction of recombinant Strain BL21 (DE 3)/pET-15 b-2423ectABC
(1) Construction of recombinant expression vectors
Using pET-15b plasmid as template, and using primer 15b-F/R to amplify to obtain linearization fragment of pET-15 b; the FL-2423 genome DNA is used as a template, a primer 15b-2423ectABC-F/R is used for amplification to obtain a tetrahydropyrimidine gene cluster of FL-2423 containing a pET-15b vector homology arm, a homologous recombinase is used for connecting a linearization fragment of pET-15b and the tetrahydropyrimidine gene cluster of FL-2423, and the gene cluster is transformed into escherichia coli DH5 alpha, ampicillin resistance screening and plasmid extraction are carried out to obtain a recombinant expression vector pET-15b-2423ectABC.
(2) Construction of recombinant strain of tetrahydropyrimidine
The recombinant expression vector pET-15b-2423ectABC is transformed into competent cells of escherichia coli BL21 (DE 3) by a thermal shock method (42 ℃ and 90 s) to obtain recombinant strain BL21 (DE 3)/pET-15 b-2423ectABC.
EXAMPLE 4 construction of recombinant Strain BL21 (DE 3)/pET-28 a-2423ectABC
The construction method is the same as in example 3 except that the plasmid used in step (1) is pET-28a, the linearization primer is 28a-F/R, the tetrahydropyrimidine gene cluster amplification primer is 28a-2423ectABC-F/R, and the selection of kana resistance is performed.
EXAMPLE 5 construction of recombinant Strain BL21 (DE 3)/pET-15 b-2423ectABC mut
(1) Construction of recombinant expression vectors
Using pET-15b plasmid as template, and using primer 15b-F/R to amplify to obtain linearization fragment of pET-15 b; the FL-2423-ectABC mut was used as a template, and primer 15b-2423ectABC-F/R mut was used to amplify to obtain the ectABC mut tetrahydropyrimidine gene cluster containing the homology arm of pET-15b vector. And (3) connecting the linearized fragment of pET-15b and ectABC mut tetrahydropyrimidine gene cluster by using homologous recombinase, transforming escherichia coli DH5 alpha, screening ampicillin resistance, and extracting plasmids to obtain a recombinant expression vector pET-15b-242 2423ectABC mut.
(2) Construction of recombinant strain of tetrahydropyrimidine
The expression vector pET-15b-2423ectABC mut was transformed into competent cells of E.coli BL21 (DE 3) by heat shock (42 ℃ C., 90 s) to obtain recombinant strain BL21 (DE 3)/pET-15 b-2423ectABC mut.
EXAMPLE 6 construction of recombinant Strain BL21 (DE 3)/pET-28 a-2423ectABC mut
The construction method is the same as in example 5 except that the plasmid used in step (1) is pET-28a, the linearization primer is 28a-F/R, the tetrahydropyrimidine gene cluster amplification primer is 28a-2423ectABC-F/R mut, and the selection of kana resistance is performed.
EXAMPLE 7 construction of recombinant Strain BL21 (DE 3)/(pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd)
(1) Construction of recombinant expression vector pETDuet-ectABC mut -lysC
Using pETDuet-1 plasmid as a template, and amplifying by using a primer pETDuet-F1/R1 to obtain a linearization fragment of pETDuet-1; the FL-2423-ectABC mut is used as a template, and a primer pETDuet-2423ectABC mut -F/R is used for amplification to obtain a ectABC mut tetrahydropyrimidine gene cluster containing a pETDuet-1 carrier homology arm. And (3) connecting the linearized fragment of pETDuet-1 and the ectABC mut tetrahydropyrimidine gene cluster by using homologous recombinase, transforming escherichia coli DH5 alpha, screening the resistance of the ampicillin, and extracting plasmids to obtain a recombinant expression vector pETDuet-ectABC mut.
Using pETDuet-ectABC mut plasmid as template, using primer pETDuet-F2/R2 to make amplification so as to obtain the linearization fragment of pETDuet-ectABC mut; the genome of corynebacterium glutamicum CGMCC 1.1886 is used as a template, and primers pETDuet-lysC-F1/R1 and pETDuet-lysC-F2/R2 are used for amplification to obtain a lysC gene fragment containing a pETDuet-ectABC mut vector homology arm, wherein the nucleic acid sequence is shown as SEQ ID NO. 10. And (3) connecting the linearized fragment of pETDuet-ectABC mut and the gene fragment of lysC by using homologous recombinase, transforming escherichia coli DH5 alpha, screening for ampicillin resistance, and extracting plasmids to obtain a recombinant expression vector pETDuet-ectABC mut -lysC.
(2) Construction of recombinant expression vector pACYCDuet-ppc-asd
Using pACYCDuet-1 plasmid as a template, and amplifying by using a primer pACYCDuet-F1/R1 to obtain a linearization fragment of pACYCDuet-1; the BL21 (DE 3) genome is used as a template, and a primer pACYCDuet-ppc-F/R is used for amplification to obtain the ppc gene containing a pACYCDuet-1 vector homology arm, and the nucleic acid sequence is shown as SEQ ID NO. 11. And (3) connecting the linearization fragment of pACYCDuet-1 and the ppc gene fragment by using homologous recombinase, transforming escherichia coli DH5 alpha, screening chloramphenicol resistance, and extracting plasmids to obtain a recombinant expression vector pACYCDuet-ppc.
Amplifying by using pACYCDuet-ppc plasmid as a template and using a primer pACYCDuet-F2/R2 to obtain a pACYCDuet-ppc linearization fragment; BL21 (DE 3) genome is used as a template, and a primer pACYCDuet-asd-F/R is used for amplification to obtain asd gene containing pACYCDuet-ppc carrier homology arm, and the nucleic acid sequence is shown as SEQ ID NO. 12. And (3) connecting the linear fragment pACYCDuet-ppc and the asd gene fragment by using homologous recombinant enzyme, transforming escherichia coli DH5 alpha, screening chloramphenicol resistance, and extracting plasmids to obtain a recombinant expression vector pACYCDuet-ppc-asd.
(3) Construction of recombinant strain of tetrahydropyrimidine
The expression vector pETDuet-ectABC mut -lysC was transformed into competent cells of E.coli BL21 (DE 3) by heat shock (42 ℃ C., 90 s) to obtain recombinant strain BL21 (DE 3)/pETDuet-ectABC mut -lysC.
The expression vector pACYCDuet-ppc-asd was transformed into E.coli BL21 (DE 3)/pETDuet-ectABC mut -lysC competent cells by heat shock (42 ℃ C., 90 s), and ampicillin and chloramphenicol resistance selection was performed to obtain recombinant strain BL21 (DE 3)/(pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd).
SEQ ID NO.10：Corynebacterium glutamicum LysC
atggccctggtcgtacagaaatatggcggttcctcgcttgagagtgcggaacgcattagaaacgtcgctgaacggatcgttgccaccaagaaggctggaaatgatgtcgtggttgtctgctccgcaatgggagacaccacggatgaacttctagaacttgcagcggcagtgaatcccgttccgccagctcgtgaaatggatatgctcctgactgctggtgagcgtatttctaacgctctcgtcgccatggctattgagtcccttggcgcagaagcccaatctttcacgggctctcaggctggtgtgctcaccaccgagcgccacggaaacgcacgcattgttgatgtcactccaggtcgtgtgcgtgaagcactcgatgagggcaagatctgcattgttgctggtttccagggtgttaataaagaaacccgcgatgtcaccacgttgggtcgtggtggttctgacaccactgcagttgcgttggcagctgctttgaacgctgatgtgtgtgagatttactcggacgttgacggtgtgtataccgctgacccgcgcatcgttcctaatgcacagaagctggaaaagctcagcttcgaagaaatgctggaacttgctgctgttggctccaagattttggtgctgcgcagtgttgaatacgctcgtgcattcaatgtgccacttcgcgtacgctcgtcttatagtaatgatcccggcactttgattgccggctctatggaggatattcctgtggaagaagcagtccttaccggtgtcgcaaccgacaagtccgaagccaaagtaaccgttctgggtatttccgataagccaggcgaggctgcgaaggttttccgtgcgttggctgatgcagaaatcaacattgacatggttctgcagaacgtctcttctgtagaagacggcaccaccgacatcatcttcacctgccctcgttccgacggccgccgcgcgatggagatcttgaagaagcttcaggttcagggcaactggaccaatgtgctttacgacgaccaggtcggcaaagtctccctcgtgggtgctggcatgaagtctcacccaggtgttaccgcagagttcatggaagctctgcgcgatgtcaacgtgaacatcgaattgatttccacctctgagattcgtatttccgtgctgatccgtgaagatgatctggatgctgctgcacgtgcattgcatgagcagttccagctgggcggcgaagacgaagccgtcgtttatgcaggcaccggacgctaa
SEQ ID NO.11：Escherichia coli ppc
atgaacgaacaatattccgcattgcgtagtaatgtcagtatgctcggcaaagtgctgggagaaaccatcaaggatgcgttgggagaacacattcttgaacgcgtagaaactatccgtaagttgtcgaaatcttcacgcgctggcaatgatgctaaccgccaggagttgctcaccaccttacaaaatttgtcgaacgacgagctgctgcccgttgcgcgtgcgtttagtcagttcctgaacctggccaacaccgccgagcaataccacagcatttcgccgaaaggcgaagctgccagcaacccggaagtgatcgcccgcaccctgcgtaaactgaaaaaccagccggaactgagcgaagacaccatcaaaaaagcagtggaatcgctgtcgctggaactggtcctcacggctcacccaaccgaaattacccgtcgtacactgatccacaaaatggtggaagtgaacgcctgtttaaaacagctcgataacaaagatatcgctgactacgaacacaaccagctgatgcgtcgcctgcgccagttgatcgcccagtcatggcataccgatgaaatccgtaagctgcgtccaagcccggtagatgaagccaaatggggctttgccgtagtggaaaacagcctgtggcaaggcgtaccaaattacctgcgcgaactgaacgaacaactggaagagaacctcggctacaaactgcccgtcgaatttgttccggtccgttttacttcgtggatgggcggcgaccgcgacggcaacccgaacgtcactgccgatatcacccgccacgtcctgctactcagccgctggaaagccaccgatttgttcctgaaagatattcaggtgctggtttctgaactgtcgatggttgaagcgacccctgaactgctggcgctggttggcgaagaaggtgccgcagaaccgtatcgctatctgatgaaaaacctgcgttctcgcctgatggcgacacaggcatggctggaagcgcgcctgaaaggcgaagaactgccaaaaccagaaggcctgctgacacaaaacgaagaactgtgggaaccgctctacgcttgctaccagtcacttcaggcgtgtggcatgggtattatcgccaacggcgatctgctcgacaccctgcgccgcgtgaaatgtttcggcgtaccgctggtccgtattgatatccgtcaggagagcacgcgtcataccgaagcgctgggcgagctgacccgctacctcggtatcggcgactacgaaagctggtcagaggccgacaaacaggcgttcctgatccgcgaactgaactccaaacgtccgcttctgccgcgcaactggcaaccaagcgccgaaacgcgcgaagtgctcgatacctgccaggtgattgccgaagcaccgcaaggctccattgccgcctacgtgatctcgatggcgaaaacgccgtccgacgtactggctgtccacctgctgctgaaagaagcgggtatcgggtttgcgatgccggttgctccgctgtttgaaaccctcgatgatctgaacaacgccaacgatgtcatgacccagctgctcaatattgactggtatcgtggcctgattcagggcaaacagatggtgatgattggctattccgactcagcaaaagatgcgggagtgatggcagcttcctgggcgcaatatcaggcacaggatgcattaatcaaaacctgcgaaaaagcgggtattgagctgacgttgttccacggtcgcggcggttccattggtcgcggcggcgcacctgctcatgcggcgctgctgtcacaaccgccaggaagcctgaaaggcggcctgcgcgtaaccgaacagggcgagatgatccgctttaaatatggtctgccagaaatcaccgtcagcagcctgtcgctttataccggggcgattctggaagccaacctgctgccaccgccggagccgaaagagagctggcgtcgcattatggatgaactgtcagtcatctcctgcgatgtctaccgcggctacgtacgtgaaaacaaagattttgtgccttacttccgctccgctacgccggaacaagaactgggcaaactgccgttgggttcacgtccggcgaaacgtcgcccaaccggcggcgtcgagtcactacgcgccattccgtggatcttcgcctggacgcaaaaccgtctgatgctccccgcctggctgggtgcaggtacggcgctgcaaaaagtggtcgaagacggcaaacagagcgagctggaggctatgtgccgcgattggccattcttctcgacgcgtctcggcatgctggagatggtcttcgccaaagcagacctgtggctggcggaatactatgaccaacgcctggtagacaaagcactgtggccgttaggtaaagagttacgcaacctgcaagaagaagacatcaaagtggtgctggcgattgccaacgattcccatctgatggccgatctgccgtggattgcagagtctattcagctacggaatatttacaccgacccgctgaacgtattgcaggccgagttgctgcaccgctcccgccaggcagaaaaagaaggccaggaaccggatcctcgcgtcgaacaagcgttaatggtcactattgccgggattgcggcaggtatgcgtaataccggctaa
SEQ ID NO.12：Escherichia coli asd
atgaaaaatgttggttttatcggctggcgcggtatggtcggctccgttctcatgcaacgcatggttgaagagcgcgacttcgacgccattcgccctgtcttcttttctacttctcagcttggccaggctgcgccgtcttttggcggaaccactggcacacttcaggatgcctttgatctggaggcgctaaaggccctcgatatcattgtgacctgtcagggcggcgattataccaacgaaatctatccaaagcttcgtgaaagcggatggcaaggttactggattgacgcagcatcgtctctgcgcatgaaagatgacgccatcatcattcttgaccccgtcaatcaggacgtcattaccgacggattaaataatggcatcaggacttttgttggcggtaactgtaccgtaagcctgatgttgatgtcgttgggtggtttattcgccaatgatcttgttgattgggtgtccgttgcaacctaccaggccgcttccggcggtggtgcgcgacatatgcgtgagttattaacccagatgggccatctgtatggccatgtggcagatgaactcgcgaccccgtcctctgctattctcgatatcgaacgcaaagtcacaaccttaacccgtagcggtgagctgccggtggataactttggcgtgccgctggcgggtagcctgattccgtggatcgacaaacagctcgataacggtcagagccgcgaagagtggaaagggcaggcggaaaccaacaagatcctcaacacatcttccgtaattccggtagatggtttatgtgtgcgtgtcggggcattgcgctgccacagccaggcattcactattaaattgaaaaaagatgtgtctattccgaccgtggaagaactgctggctgcgcacaatccgtgggcgaaagtcgttccgaacgatcgggaaatcactatgcgtgagctaaccccagctgccgttaccggcacgctgaccacgccggtaggccgcctgcgtaagctgaatatgggaccagagttcctgtcagcctttaccgtgggcgaccagctgctgtggggggccgcggagccgctgcgtcggatgcttcgtcaactggcgtaa
Example 8 fermentation production of tetrahydropyrimidine Using glucose as substrate
(1) Flat seed activation
The recombinant strain is streaked on an eggplant-shaped bottle containing a resistant solid LB culture medium in an ultra-clean workbench, and is cultivated for 12 hours at a constant temperature of 37 ℃ to obtain activated seeds.
(2) Seed liquid preparation
The activated seeds in the eggplant-shaped bottle are inoculated into a fermentation tank filled with 10L of seed culture medium, the temperature is 37 ℃, dissolved oxygen is 20-30%, ammonia water is used for regulating and controlling the pH value to 7.0, and the seeds are cultured until OD=15 to obtain seed liquid.
The seed culture medium comprises the following components: 30.0g/L glucose, 1.5g/L peptone, 6.8g/L yeast powder, 0.68g/L citric acid monohydrate, 1.6g/L potassium dihydrogen phosphate, 0.68g/L magnesium sulfate heptahydrate and 0.33g/L GPE defoamer.
(3) Fermentation culture
Inoculating the seed liquid into a fermentation tank filled with 8L of fermentation medium according to the transfer amount of 15%, regulating the pH to 7.0 by using ammonia water at 37 ℃, adding IPTG to the fermentation medium until the concentration is 0.1mM/L after the bottom sugar is consumed, regulating the growth rate of OD to 3/h by using glucose solution with the concentration of 700g/L, regulating the pH to 7.4 by using ammonia water, accelerating the sugar supplementing speed to the sugar concentration of 0.5g/L when the OD grows to 55, and fermenting for 30h to obtain the fermentation liquid containing tetrahydropyrimidine.
The fermentation medium comprises the following components: 25.0g/L of glucose, 2.5g/L of peptone, 3.8g/L of yeast powder, 6.8g/L of corn steep liquor dry powder, 2.5g/L of citric acid monohydrate, 3.8g/L of potassium dihydrogen phosphate, 1.5g/L of magnesium sulfate heptahydrate and 0.85g/L of GPE defoamer.
Comparative example 1 construction of recombinant Strain BL21 (DE 3)/pET-15 b-2581ectABC
(1) Construction of recombinant expression vectors
Using pET-15b plasmid as template, and using primer 15b-F/R to amplify to obtain linearization fragment of pET-15 b; the DSM 2581 genome DNA is used as a template, primers 15b-2581ectABC-F/R are used for amplification to obtain a tetrahydropyrimidine gene cluster of DSM 2581 containing a pET-15b vector homology arm, and the nucleic acid sequence is shown as SEQ ID NO. 8. The linearized fragment of pET-15b and the tetrahydropyrimidine gene cluster of DSM 2581 are connected by homologous recombinase, escherichia coli DH5 alpha is transformed, ampicillin resistance screening is carried out, and plasmids are extracted to obtain a recombinant expression vector pET-15b-2581ectABC.
(2) Construction of recombinant strains
The expression vector pET-15b-2581ectABC was transformed into competent cells of E.coli BL21 (DE 3) by heat shock (42 ℃ C., 90 s) to obtain recombinant strain BL21 (DE 3)/pET-15 b-2581ectABC.
SEQ ID NO.9: h.elongate DSM 2581 tetrahydropyrimidine Gene Cluster
atgaacgcaaccacagagccctttacaccctccgccgacctggccaagcccagcgtggccgatgccgtggtcggccatgaggcctcaccgctcttcatccgcaagccaagccccgatgacggctggggcatctacgagctggtcaagtcctgtccgcctctcgacgtcaattccgcctacgcctatctgttgctggccacccagttccgcgatagctgcgccgtggcgaccaacgaagagggcgagatcgtcggcttcgtttccggctacgtgaagagcaacgcccccgatacctatttcctctggcaggttgccgtgggcgagaaggcacgtggcaccggcctggcccgtcgtctggtggaagccgtgatgacacgcccggaaatggccgaggtccaccatctcgagaccactatcacgcccgacaaccaggcgtcctggggcttgttccgccgtctcgccgatcgctggcaggcgccgttgaacagccgcgaatacttctccaccgatcaactcggcggtgagcatgacccggaaaacctcgttcgcatcggcccgttccagaccgaccagatctgagccgggacgccgcctggccggcccggtacgggccggcaacccgtcttttcgttttatcactttccccccacaggaggtcgcaatgcagacccagattctcgaacgcatggagtccgacgttcggacctactcccgctccttcccggtcgtcttcaccaaggcgcgcaatgcccgcctgaccgacgaggaagggcgcgagtacatcgacttcctggccggtgccggcaccctgaactacggccacaacaacccgcacctcaagcaggcgctgctcgactatatcgacagcgacggcatcgtccacggcctggacttctggactgcggccaagcgcgactatctggaaaccctggaagaggtgatcctcaagccgcgcggtctcgactacaaggtgcatctgcccggaccgactggcaccaacgccgtcgaggcggccattcgcctggcccgggtcgccaaggggcgccacaatatcgtctccttcaccaacggctttcatggcgtcaccatgggcgcgctggcgaccaccggtaaccgcaagttccgcgaggccaccggtggcgtgccgacccaggctgcttccttcatgccgttcgatggctacctcggcagcagcaccgacaccctcgactacttcgagaagctgctcggcgacaagtccggcggcctggacgtgcccgcggcggtgatcgtcgagacagtgcagggcgagggcggtatcaatgtcgccggcctggagtggctcaagcgcctcgagagcatctgccgcgccaatgacatcctgctgatcatcgacgacatccaggcgggctgcggccggaccggcaagttcttcagcttcgagcatgccggcatcacgccggatatcgtgaccaactccaagtcgctgtccggttacggcctgccgttcgctcacgtcctgatgcgccccgagctcgacaagtggaagcccggtcagtacaacggcaccttccgcggcttcaacctggctttcgccactgctgctgccgccatgcgcaagtactggagcgacgacaccttcgagcgtgacgtgcagcgcaaggctcgcatcgtcgaggaacgcttcggcaagatcgccgcctggctgagcgagaacggcatcgaggcctccgagcgcggccgcgggctgatgcggggcatcgacgtgggttccggcgatatcgccgacaagatcacccaccaagccttcgagaacgggttgatcatcgaaaccagcggtcaggacggcgaagtggtcaagtgcctgtgcccgctgaccattcccgacgaagacctggtcgagggactcgacatcctcgagaccagcaccaagcaggcctttagctgatcgcctgaggtgcgccatcgggcctgtccatggcatcctgtatcggtcggccgtgcgcggccggccagtcattgattcactggagaatcgacatgatcgttcgcaatctcgaagaagcgcgccagaccgaccgtctggtcaccgccgaaaacggcaactgggacagcacccgcctgtcgctggccgaagatggtggcaactgctccttccacatcacccgcatcttcgagggtaccgagacccacatccactataagcatcacttcgaggctgtttattgcatcgaaggcgagggcgaagtggaaaccctggccgatggcaagatctggcccatcaagccgggtgacatctacatcctcgaccagcacgacgagcacctgctgcgcgccagcaagaccatgcacctggcctgcgtgttcacgccgggcctgaccggcaacgaagtgcaccgcgaagacggttcctacgcacctgccgacgaagccgacgaccagaagccgctgtaa
Construction of comparative example 2 recombinant Strain BL21 (DE 3)/pET-28 a-2581ectABC
The construction method is the same as that of comparative example 1, except that the plasmid used in the step (1) is pET-28a, the linearization primer is 28a-F/R, and the tetrahydropyrimidine gene cluster amplification primer is 28 a-2581-ectABC-F/R, and the selection of the kana resistance is performed.
The primer sequences used above are detailed in Table 1.
TABLE 1 primer sequences
Experimental example 1
The recombinant strains of comparative example 1, example 3, example 5 and example 7 were fermented under the same conditions as in example 8 to obtain a fermentation broth containing tetrahydropyrimidine, and the synthesis efficiency of tetrahydropyrimidine was measured. The measurement results are shown in Table 2.
TABLE 2 Synthesis efficiency of tetrahydropyrimidines of different Gene clusters
As can be seen from Table 2, the recombinant strain of comparative example 1 had a tetrahydropyrimidine fermentation content of 5.6g/L, the recombinant strain of example 3 had a tetrahydropyrimidine fermentation content of 6.8g/L, the recombinant strain of example 5 had a tetrahydropyrimidine fermentation content of 9.2g/L, and the recombinant strain of example 7 had a tetrahydropyrimidine fermentation content of 75.6g/L. The recombinant strain of example 3 had an increased tetrahydropyrimidine fermentation content of 21.4%, the recombinant strain of example 5 had an increased tetrahydropyrimidine fermentation content of 64.3% and the recombinant strain of example 7 had a fermentation content of 13.5 times that of the recombinant strain of comparative example 1, as compared to the recombinant strain of comparative example 1; compared with the recombinant strain of example 3, the tetrahydropyrimidine fermentation content of the recombinant strain of example 5 was increased by 35.3%. The result shows that the tetrahydropyrimidine gene cluster derived from the bacillus caldus has the advantages that the efficiency of synthesizing the tetrahydropyrimidine by the tetrahydropyrimidine gene cluster of the DSM 2581 strain is improved by more than 20 percent, and the synthesis efficiency after mutation is improved by more than 60 percent; compared with the original gene cluster, the efficiency of synthesizing the tetrahydropyrimidine by the tetrahydropyrimidine gene cluster mutant from the bacillus salicillus is improved by more than 30%.
Therefore, the provided tetrahydropyrimidine gene cluster and mutant derived from the bacillus halophilus are utilized for fermenting and producing the tetrahydropyrimidine, so that the fermentation content of the tetrahydropyrimidine can be improved.
Experimental example 2
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 broth containing tetrahydropyrimidine, and the fermentation content of tetrahydropyrimidine was measured. The measurement results are shown in Table 3.
TABLE 3 Synthesis efficiency of tetrahydropyrimidines of different Gene clusters
As can be seen from Table 3, the recombinant strain of comparative example 2 had a tetrahydropyrimidine fermentation content of 8.4g/L, the recombinant strain of example 4 had a tetrahydropyrimidine fermentation content of 10.8g/L, the recombinant strain of example 6 had a tetrahydropyrimidine fermentation content of 14.5g/L, and the recombinant strain of example 7 had a tetrahydropyrimidine fermentation content of 75.6g/L. The recombinant strain of example 4 had 28.6% increased tetrahydropyrimidine fermentation content, the recombinant strain of example 6 had 72.6% increased tetrahydropyrimidine fermentation content, and the recombinant strain of example 7 had 9 times the tetrahydropyrimidine fermentation content of the recombinant strain of comparative example 2, compared to the recombinant strain of comparative example 2. Compared with the recombinant strain of example 4, the fermentation content of tetrahydropyrimidine of the recombinant strain of example 6 is improved by 34.3%. The result shows that the tetrahydropyrimidine gene cluster derived from the bacillus caldus has the advantages that the efficiency of synthesizing the tetrahydropyrimidine by the tetrahydropyrimidine gene cluster of the DSM 2581 strain is improved by more than 20 percent, and the synthesis efficiency after mutation is improved by more than 70 percent; compared with the original gene cluster, the efficiency of synthesizing the tetrahydropyrimidine by the tetrahydropyrimidine gene cluster mutant from the bacillus salicillus is improved by more than 30%.
Therefore, the provided tetrahydropyrimidine gene cluster and mutant derived from the bacillus halophilus are utilized for fermenting and producing the tetrahydropyrimidine, so that the fermentation content of the tetrahydropyrimidine can be improved.
Experimental example 3
Recombinant strain BL21 (DE 3)/(pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd) was fermented in fermentation medium of different initial glucose concentrations under the same process conditions as in example 8, and the fermentation content of tetrahydropyrimidine was measured, and the measurement results are shown in Table 4.
TABLE 4 influence of different initial glucose concentrations
As is clear from Table 4, the OD at the end of the bottom glucose was 4.8, the fermentation content was 30.8g/L, the OD at the end of the bottom glucose was 9.5, the fermentation content was 54.7g/L, the OD at the end of the bottom glucose was 30.0, the fermentation content was 75.6g/L, the OD at the end of the bottom glucose was 35g/L, the fermentation content was 43.2, the fermentation content was 57.3g/L, the OD at the end of the bottom glucose was 45g/L, and the fermentation content was 42.6g/L, respectively, each of which was 5g/L, 15g/L, 25g/L, and 25 g/L. As can be seen, the fermentation content of tetrahydropyrimidine is highest at a bottom sugar concentration of 25 g/L. Because the addition of the inducer can inhibit the growth of cells, the initial glucose concentration is low, so that the OD is small during induction, the biomass is low during induction, the thalli cannot grow to a higher cell concentration, and the tetrahydropyrimidine content is low; when the initial glucose concentration is high and the biomass is too high after the bottom sugar is consumed, the thalli are in an aging state, the activity is reduced, the induction effect is reduced, and the content of tetrahydropyrimidine is low, which is called the Klebsiella effect.
The klebsiella effect is also called glucose effect, which means that the metabolic product of glucose can reduce the level of cAMP, on one hand, CAP protein of cAMP is combined with CAP binding site on DNA, RNA polymerase can be combined with the promoter region of lactose operon to start transcription; cAMP on the other hand acts as a second messenger to modulate cellular responses by activating APK (cAMP dependent protein kinase) to phosphorylate target cell proteins. Therefore, when the glucose content in the fermentation liquid is high, even if an inducer exists, the inducer can have adverse effect on induction and is unfavorable for enzyme expression, so that the induction is generally required to start after the consumption of the bottom sugar in fermentation, and the concentration of the bottom sugar is important for the synthesis of the enzyme which needs to be induced for expression.
Experimental example 4
In the fermentation process of recombinant strain BL21 (DE 3)/(pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd), the different OD growth rates were regulated after the consumption of the bottom sugar, and the fermentation content of tetrahydropyrimidine was measured under the same process conditions as in example 8, and the measurement results are shown in Table 5.
TABLE 5 influence of different OD growth rates
As is clear from Table 5, when the OD growth rate was 0 to 2/h, the fermentation content of tetrahydropyrimidine was 50.5g/L, when the OD growth rate was 2 to 4/h, the fermentation content of tetrahydropyrimidine was 75.6g/L, when the OD growth rate was 4 to 6/h, the fermentation content of tetrahydropyrimidine was 63.4g/L, and when the OD growth rate was 7 to 8/h, the fermentation content of tetrahydropyrimidine was 35.8g/L. It can be seen that the fermentation content of tetrahydropyrimidine is highest at an OD increase rate of 2-4/h. This is because after the inducer is added, the strain starts to convert from reproductive growth to the enzyme related to the synthesis of the tetrahydropyrimidine, the OD growth speed is controlled by feeding glucose, the OD growth speed is too slow, the inducer has larger damage to cells, the final biomass is lower, and the tetrahydropyrimidine content is low; the OD increases too fast, resulting in poor induction effect, less expression of the enzyme related to the tetrahydropyrimidine, and low fermentation content of the tetrahydropyrimidine. After the bottom sugar is consumed, the fed-batch glucose solution with the OD growth speed controlled to be 2-4 can balance the reproduction growth of the strain and the expression of the enzyme related to the tetrahydropyrimidine, and the fermentation content of the tetrahydropyrimidine is highest.
Experimental example 5
In the fermentation process of recombinant strain BL21 (DE 3)/(pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd), the sugar supplementing speed is respectively accelerated to 45-65 to regulate the sugar concentration to 0.2-1g/L when the OD grows, and the fermentation content of tetrahydropyrimidine is measured under other process conditions as in example 8, and the measurement results are shown in Table 6.
TABLE 6 Effect of increasing sugar feeding Rate at different OD' s
Because of the glucose effect, even if an inducer is present at a high glucose level in the fermentation broth, the induction of the expression of the tetrahydropyrimidine synthesis-related enzyme occurs at a very low glucose concentration, but the substrate for the tetrahydropyrimidine synthesis is glucose, so that the expression of the tetrahydropyrimidine-related enzyme and the synthesis of the tetrahydropyrimidine need to be regulated by regulating the glucose concentration in the fermentation broth.
As is clear from Table 6, the sugar feeding rate was increased at an OD of 45, the fermentation content of tetrahydropyrimidine was 62.4g/L, the sugar feeding rate was increased at an OD of 55, the fermentation content of tetrahydropyrimidine was 75.6g/L, and the sugar feeding rate was increased at an OD of 65, and the fermentation content of tetrahydropyrimidine was 53.7g/L. Therefore, when the glucose solution with the concentration of 700g/L is fed to the fermentation OD of 55, the sugar supplementing speed is increased, the sugar concentration is regulated to be 0.2-1g/L, and the fermentation content of the tetrahydropyrimidine is highest.
Experimental example 6
During the fermentation of recombinant strain BL21 (DE 3)/(pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd), the initial pH was adjusted and the fermentation content of tetrahydropyrimidine was determined under the same conditions as in example 8, and the measurement results are shown in Table 7.
TABLE 7 influence of different initial pH values
When the bottom sugar is consumed, the bacterial strain is active in reproduction and growth, the OD (optical density) growth rate is regulated and controlled to be 2-4/h by adding a glucose solution with the concentration of 700g/L, the fermentation pH can slowly rise by 0.3-0.5 due to insufficient sugar supply, the activity of the bacterial strain and the enzyme activity can be reduced due to excessive pH, and the fermentation content of the tetrahydropyrimidine is influenced. As is clear from Table 7, the fermentation content of tetrahydropyrimidine was 70.4g/L at an initial pH of 6.8, 75.6g/L at an initial pH of 7.0, 63.9g/L at an initial pH of 7.2, and 51.5g/L at an initial pH of 7.4. At an initial pH of 7.0, the fermentation content of tetrahydropyrimidine was highest.
Experimental example 7
During the fermentation of recombinant strain BL21 (DE 3)/(pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd), the pH after induction was controlled differently, the fermentation content of tetrahydropyrimidine was determined under the same process conditions as in example 8, and the measurement results are shown in Table 8.
TABLE 8 influence of pH after different induction
The optimal pH for culturing the escherichia coli is neutral or neutral alkali, but the enzyme activities are different under different pH conditions. As can be seen from the data in Table 8, under the condition that the initial pH is 7.0, the fermentation content of the tetrahydropyrimidine is 58.7g/L when the pH after induction is 6.8, the fermentation content of the tetrahydropyrimidine is 63.5g/L when the pH after induction is 7.0, the fermentation content of the tetrahydropyrimidine is 70.8g/L when the pH after induction is 7.2, the fermentation content of the tetrahydropyrimidine is 75.6g/L when the pH after induction is 7.4, and the enzyme activity is high and the fermentation content of the tetrahydropyrimidine is highest when the pH after induction is 7.4.
In conclusion, the recombinant strain BL21 (DE 3)/(pETDuet-ectABC mut -lysC, pACYCDuet-ppc-asd) constructed by utilizing the tetrahydropyrimidine biosynthesis gene cluster with the nucleic acid sequence shown in SEQ ID No.8 is used for fermenting and producing the tetrahydropyrimidine, and the fermentation process is controlled by optimizing the concentration of the bottom sugar, the OD (OD) growth speed and the sugar supplementing time and the fermentation pH, so that the synthesis efficiency of the tetrahydropyrimidine is improved, the fermentation content of the tetrahydropyrimidine is high, the production cost of the tetrahydropyrimidine is low, and the popularization and the application of the tetrahydropyrimidine are facilitated.
Therefore, the invention constructs the recombinant strain by using the tetrahydropyrimidine gene cluster and the mutant, improves the synthesis efficiency and the content of the tetrahydropyrimidine produced by fermentation, is beneficial to improving the yield of the tetrahydropyrimidine, reduces the production cost of the tetrahydropyrimidine, and is beneficial to popularization and utilization of the tetrahydropyrimidine.
Claims (10)
1. A bacillus salicillus-derived tetrahydropyrimidine gene cluster, characterized in that: the tetrahydropyrimidine gene cluster nucleic acid sequence is shown as SEQ ID NO.7, and comprises the following 3 genes: ectA gene, the nucleic acid sequence is shown as SEQ ID NO. 1; ectB gene, the nucleic acid sequence is shown as SEQ ID NO. 2; ectC gene and nucleic acid sequence shown in SEQ ID NO. 3.
2. The bacillus halophilus-derived tetrahydropyrimidine gene cluster according to claim 1, wherein: the tetrahydropyrimidine gene cluster is derived from bacillus caldus Halobacillus sp, FL-2423 with tetrahydropyrimidine secretion characteristics, and the strain is preserved in China general microbiological culture Collection center (CGMCC) No.18783 in 11 months 01 in 2019, and has a preservation address of China academy of sciences of China, no. 3 of the West Liu 1 in North Star in the Chao yang area of Beijing.
3. A mutant formed by a tetrahydropyrimidine gene cluster derived from bacillus salicillus, characterized in that: the nucleic acid sequence of the mutant is shown as SEQ ID NO. 8.
4. A mutant of a tetrahydropyrimidine gene cluster derived from bacillus salicillus as claimed in claim 3, wherein: the mutant consists of amino acid sequences shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.
5. The mutant of the bacillus halophilus-derived tetrahydropyrimidine gene cluster according to claim 4, wherein: the amino acid sequence shown in SEQ ID NO.4 is a ectA gene original sequence shown in SEQ ID NO.1, and has the following mutation: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is replaced by I from L at the 54 th amino acid and by T from Q at the 103 th amino acid.
6. The mutant of the bacillus halophilus-derived tetrahydropyrimidine gene cluster according to claim 4, wherein: the amino acid sequence shown in SEQ ID NO.5 is a ectB gene original sequence with a nucleic acid sequence shown in SEQ ID NO.2, and has the following mutation: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is replaced by Q at the 28 th amino acid, P at the 67 th amino acid, T at the 189 th amino acid, H at the 251 nd amino acid, and E at the 331 st amino acid.
7. The mutant of the bacillus halophilus-derived tetrahydropyrimidine gene cluster according to claim 4, wherein: the amino acid sequence shown in SEQ ID NO.6 is a ectC gene original sequence shown in SEQ ID NO.3, and has the following mutation: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is replaced by E from A at the 46 th amino acid, R from D at the 90 th amino acid and E from Q at the 134 th amino acid.
8. The application of a bacillus salicillus-derived tetrahydropyrimidine gene cluster and a mutant formed by the same in the process of synthesizing tetrahydropyrimidine is characterized in that: the method comprises the following steps:
(1) Obtaining a bacillus salicillus FL-2423 tetrahydropyrimidine gene cluster;
(2) Preparing bacillus salicillus FL-2423 tetrahydropyrimidine gene cluster mutants;
(3) Constructing a recombinant vector containing FL-2423 tetrahydropyrimidine gene cluster or a tetrahydropyrimidine gene cluster mutant;
(4) Constructing a tetrahydropyrimidine recombinant strain;
(5) And fermenting with glucose as a substrate to produce the tetrahydropyrimidine.
9. The use of a bacillus halophilus-derived tetrahydropyrimidine gene cluster and mutants thereof in the synthesis of tetrahydropyrimidine according to claim 8, wherein: the step (5) comprises seed plate activation, seed liquid preparation and fermentation production of the tetrahydropyrimidine, wherein the specific operation of the fermentation production of the tetrahydropyrimidine is as follows: the seed liquid is put into a fermentation tank filled with 8L of fermentation medium, the temperature is 37 ℃, dissolved oxygen is 20-30%, ammonia water is used for regulating and controlling the initial pH value to 7.0, IPTG is added to the fermentation medium until the concentration is 0.1mM/L after the bottom sugar is consumed, glucose solution with the concentration of 700g/L is fed, the growth rate of OD is regulated and controlled to be 2-6/h, ammonia water is added for regulating and controlling the pH value to 7.4, the sugar supplementing speed is accelerated until the sugar concentration is 0.2-1g/L when the OD grows to 55, and fermentation is finished for 30h, so that the fermentation liquid containing tetrahydropyrimidine is obtained.
10. The use of a bacillus halophilus-derived tetrahydropyrimidine gene cluster and mutants thereof in the synthesis of tetrahydropyrimidine according to claim 9, wherein: and after the bottom sugar is consumed, adding IPTG to the fermentation medium until the concentration is 0.1mM/L, feeding glucose solution with the concentration of 700g/L, and regulating the OD growth rate to be 2-4/h.
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