CN114621968A

CN114621968A - Tetrahydropyrimidine biosynthesis gene cluster, mutant and method for preparing tetrahydropyrimidine

Info

Publication number: CN114621968A
Application number: CN202210531189.1A
Authority: CN
Inventors: 叶健文; 周明新
Original assignee: Shenzhen Zhongkeling Carbon Biotechnology Co ltd
Current assignee: Shenzhen Zhongkeling Carbon Biotechnology Co ltd
Priority date: 2022-05-17
Filing date: 2022-05-17
Publication date: 2022-06-14
Anticipated expiration: 2042-05-17
Also published as: CN114621968B

Abstract

The invention provides a biosynthetic gene cluster of tetrahydropyrimidine, which is used for producing and re-screening through recombinant expression fermentation to obtain recombinant escherichia coli capable of stably producing tetrahydropyrimidine at a high yield by using aspartic acid. The yield of the gene cluster before mutation under the same fermentation condition is increased by about 30%, and the efficiency is further improved by about 20% through mutation.

Description

Tetrahydropyrimidine biosynthesis gene cluster, mutant and method for preparing tetrahydropyrimidine

Technical Field

The invention relates to the technical field of synthetic biology, in particular to a tetrahydropyrimidine biosynthesis gene cluster and a method for preparing tetrahydropyrimidine.

Background

Halophilic bacteria belong to an extreme microorganism and can be classified into mild halophilic bacteria (the optimum NaCl concentration is 10-30 g/L), moderate halophilic bacteria (the optimum NaCl concentration is 50-100 g/L) and extreme halophilic bacteria (the optimum NaCl concentration is 130-. In order to maintain the osmotic pressure balance inside and outside the cell, some substances are accumulated in the halophilic bacteria cell to resist the external hypertonic environment, and the substances mainly comprise amino acids and derivatives thereof, polyhydric alcohols, sugars and tetrahydropyrimidine (Ectoine,1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid), wherein the tetrahydropyrimidine has important application value.

In 1985 Galinski et al were first extremely halophilicHalorhodospira halochlorsThe Ectoine has the molecular weight of 142.16, is extremely hydrophilic, belongs to organic small molecules with zwitterion characteristics, and has the structural formula shown in the specification.

The large accumulation of tetrahydropyrimidine in cells can resist the impact of high osmotic pressure, and can stabilize the hydration layer structure of protein (especially under the conditions of freezing, drying and high temperature). Recent studies show that tetrahydropyrimidine has a stabilizing effect on macromolecules such as enzymes, DNA and membranes in vivo and in vitro; can prevent skin dehydration and dryness while reducing damage degree of ultraviolet rays to skin, and in addition, the tetrahydropyrimidine can inhibit formation of amyloid protein and can reduce the initial and elongation stages of the formation of the amyloid protein, thereby having a potential function of preventing senile dementia.

Because tetrahydropyrimidine has a chiral carbon atom in a molecule, the tetrahydropyrimidine is difficult to synthesize by a chemical method, and the existing production method is mainly a microbial fermentation method. Currently, the model strains widely used in the commercial scale production of Ectoine are mainly concentrated on the family Halomonas (of the class of γ -Proteobacteria: (A)Halomonadaceae) Especially of the genus Halomonas (Halomonas) There are many. After decades of researchThe synthetic route of tetrahydropyrimidine has been developed more deeply at the gene level, enzyme level and regulation level.HalomonasAs a model population for the tetrahydropyrimidine synthesis study, reportsH.elongate DSM 2581、H.elongate DSM 3043、H. elongateThe anabolic pathway of Ectoine in ATCC 33174. In the above-mentioned anabolic pathway of tetrahydropyrimidine, aspartate semialdehyde (L-aspartate-B-semialdehyde) in the biosynthetic pathway of L-lysine is used as a precursor, and the linkage which is highly conserved by evolution is relied onectABCThe gene cluster operon (ect-operon). Structural geneectB、ectAAndectCrespectively encoding L-diaminobutyric acid transaminase: (A)ectB) L-diaminobutyric acid acetyltransferase (A), (B), (C) and C)ectA) And tetrahydropyridine synthase (ectC) Synthesizing tetrahydropyrimidine through 3 steps of catalysis.

The existing metabolic pathway is adopted to synthesize the tetrahydropyrimidine, so that the defect of low yield is caused, the large-scale industrial application of the tetrahydropyrimidine is limited, and the method for improving the yield of the tetrahydropyrimidine at high yield of the aspartic acid becomes the key point of industrial research.

The inventor utilizes the method of carrying out the macro genome sequencing analysis after the salt lake sludge is sampled and obtaining a plurality of brand new sequences through sequence comparison and screeningectABCGene clusters are compared and searched by BLAST based on NCBI database,ectABCthe nucleotide sequence is obviously different from other halomonas, recombinant Escherichia coli capable of utilizing aspartic acid to stably produce high-yield tetrahydropyrimidine is obtained by recombinant expression fermentation production rescreening, and then the obtained product is subjected toectABCThe gene cluster is subjected to mutation design, so that the activity is enhanced, and the conversion efficiency of tetrahydropyrimidine is further improved.

Disclosure of Invention

The invention aims to solve the technical problem of providing a gene cluster for synthesizing tetrahydropyrimidineectABCThe method can be used for efficiently synthesizing tetrahydropyrimidine, and the yield of the tetrahydropyrimidine is greatly improved.

Based on the above, the invention provides a tetrahydropyrimidine biosynthesis gene cluster, which at least comprises 3 genes, wherein each gene is as follows:

ectAthe gene has a nucleic acid sequence shown as SEQ ID NO. 1;

ectBthe gene and the nucleic acid sequence are shown as SEQ ID NO. 2;

ectCthe gene and the nucleic acid sequence are shown as SEQ ID NO. 3.

The gene cluster is derived from Halomonas sp.YL 01, which is preserved in Guangdong provincial microorganism culture Collection (GDMCC), address: building 5 of first furnance, large yard, 100, building 59, Guangdong province, Guangzhou, China, zip code: 510070, the preservation date is 24/4/2022, and the preservation number is GDMCC No. 62420.

The invention also provides a method for acquiring the biosynthesis gene cluster of tetrahydropyrimidine,

firstly, screening strains, namely screening the strains contained in the salt lake sludge;

secondly, PCR amplification verification;

and thirdly, sequencing the genome to obtain a gene cluster.

The invention also provides a tetrahydropyrimidine biosynthesis gene cluster mutant and a preparation method thereofectAThe gene and the nucleic acid sequence have the following mutations on the basis of the original sequence shown in SEQ ID NO. 1: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is shown in SEQ ID NO.6 after mutation, wherein the amino acid at the 39 th position is replaced by N to S, and the amino acid at the 132 th position is replaced by A to T.

It is composed ofectBThe gene has the following mutations on the basis of the original sequence shown in SEQ ID NO. 2: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is changed from I to L at the 59 th amino acid, from N to G at the 71 th amino acid, from A to T at the 179 th amino acid and from V to I at the 381 th amino acid, and the amino acid sequence of the polypeptide after mutation is shown as SEQ ID NO. 8.

It is composed ofectCThe gene and the nucleic acid sequence have the following mutations on the basis of the original sequence shown in SEQ ID NO. 3: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is replaced by Y from F at the 62 th amino acid, and the mutated amino acid sequence of the polypeptide is shown as SEQ ID NO. 10.

The construction method of the synthetic gene cluster mutant comprises the following steps:

firstly, extracting a chassis bacteria gene cluster;

secondly, expressing the vector skeleton and performing PCR amplification on the extracted gene cluster;

thirdly, constructing recombinant expression plasmids;

and fourthly, constructing a mutant.

Expression vector or recombinant microorganism engineering bacteria containing the synthetic gene cluster or gene cluster mutant.

The gene cluster, the gene cluster mutant, the expression vector and the recombinant microorganism engineering bacteria are applied to the process of synthesizing the tetrahydropyrimidine.

The invention also provides a method for producing tetrahydropyrimidine by adopting the recombinant microorganism engineering bacteria, which converts glucose into tetrahydropyrimidine through whole cells.

The method further comprises:

firstly, preparing seed liquid;

step two, tetrahydropyrimidine fermentation;

and thirdly, preparing tetrahydropyrimidine.

The invention has the following beneficial technical effects: the invention provides a gene cluster for synthesizing tetrahydropyrimidineectABCAnd mutants thereof, which can be used for efficiently synthesizing tetrahydropyrimidine, and research shows that the gene clusterectABCThe catalytic efficiency of the coded enzyme is better, the yield is increased by about 30% under the same fermentation condition, and meanwhile, the efficiency is further improved by about 20% by mutating the gene cluster.

Drawings

FIG. 1 is a liquid phase mass spectrometry analysis of tetrahydropyrimidine production by recombinant E.coli;

FIG. 2 shows the present inventionectAGenes andHalomonas aestuarii Hb3、Halomonas sp. THAF5a、Halomonas sp. JS92-SW72、Halomonas sp. BM-2019、Halomonas elongatainectAComparing the gene sequences;

FIGS. 3 and 4 illustrate the present inventionectBGenes andHalomonas aestuarii Hb3、Halomonas sp.THAF5a、Halomonas sp. JS92-SW72、Halomonas sp. BM-2019、Halomonas elongatainectBThe gene sequence alignment results, wherein FIG. 4 is the alignment of FIG. 3Continuing;

FIG. 5 shows the present inventionectCGenes andHalomonas aestuarii Hb3、Halomonas sp. THAF5a、Halomonas sp. JS92-SW72、Halomonas sp. BM-2019、Halomonas elongatainectCAnd (5) gene sequence comparison results.

FIG. 6 shows a gene cluster encoding a key enzyme for synthesizing high-activity tetrahydropyrimidineectABCSchematic representation of the recombinant expression map of (1).

FIG. 7 is a drawing showingectABCPcr products of the gene cluster.

Detailed Description

The invention provides a biosynthesis gene cluster of tetrahydropyrimidine, which at least comprises 3 genes which are respectively as follows:

ectAthe gene has a nucleic acid sequence shown as SEQ ID NO. 1;

ectBthe gene and the nucleic acid sequence are shown as SEQ ID NO. 2;

ectCthe gene and the nucleic acid sequence are shown as SEQ ID NO. 3.

The gene cluster is derived from Halomonas sp.YL 01, which is preserved in Guangdong provincial microorganism culture Collection (GDMCC), address: building 5 of first furios middle way 100 large yard 59, Guangdong province, Guangzhou, zip code: 510070, the preservation date is 24/4/2022, and the preservation number is GDMCC No. 62420.

secondly, PCR amplification verification;

and thirdly, sequencing the genome to obtain a gene cluster.

The invention also provides a tetrahydropyrimidine biosynthesis gene cluster mutant and a preparation method thereofectAThe gene and the nucleic acid sequence have the following mutations on the basis of the original sequence shown in SEQ ID NO. 1: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is shown in SEQ ID NO.4 after mutation, wherein the amino acid at the 39 th position is replaced by N to S, and the amino acid at the 132 th position is replaced by A to T.

It is composed ofectBThe gene and the nucleic acid sequence have the following mutations on the basis of the original sequence shown in SEQ ID NO. 2: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is changed from I to L at the 59 th amino acid, from N to G at the 71 th amino acid, from A to T at the 179 th amino acid and from V to I at the 381 th amino acid, and the amino acid sequence of the polypeptide after mutation is shown as SEQ ID NO. 5.

Its ectCThe gene has the following mutations on the basis of the original sequence shown in SEQ ID NO.3 as the nucleic acid sequence: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is replaced by Y from F at the 62 th amino acid, and the mutated amino acid sequence of the polypeptide is shown as SEQ ID NO. 6.

SEQ ID NO.1：ectA(as shown in sequence 1 in the sequence table)

ATGACAATGAACGCAACCACCGAGCCCTTCACACCCTCCGCCGACCTGGCACGCCCCACCGTGGCGGACGCCGTGGTCGGTCACGAGGCCTATCCGCTGTTCATCCGCAAGCCCAACCCCGATGACGGCTGGGGCATCTACGAGCTGGTCAAGTCCTGCCCCCCGCTGGACGTCAACTCCGCCTATGCCTACCTGCTGCTGGCGACCCAGTTCCGCGACAGTTGTGCCGTGGCCACCAACGAGGAGGGCGAGATCGTCGGTTTCGTCTCCGGCTACGTGAAGAGCAACGCCCCGGACACCTACTTCCTGTGGCAGGTGGCGGTCGGCGAGAAGGCGCGCGGCACCGGCCTGGCCCGGCGCCTGGTGGAAGCCGTGATGACCCGCCCGGAGATGGCCGAGGTCCACCACCTCGAGACCACCATCACCCCCGACAACCAGGCCTCCTGGGGCCTGTTCCGGCGGCTTGCCGAACGCTGGCAGGCGCCGCTCAACAGCCGCGAGTACTTCTCCACCGACCAGCTCGGTGGCGAGCACGACCCGGAAAACCTCGTGCGCATCGGCCCCTTCCAGACCGATCGCATCTGA

SEQ ID NO.2：ectB (as shown in sequence 2 in the sequence table)

ATGCAGACCCAGATCCTCGAACGCATGGAGTCCGAAGTTCGGACCTATTCCCGCTCCTTTCCGGTGGTCTTCACCAAGGCCCGGAATGCCCGTCTGACCGACGAGGACGGCCGCGAGTACATCGACTTCCTGGCCGGTGCCGGCACCCTGAACTACGGCCACAACAACCCGCACATCAAGCAGGCGCTGCTCGACTACCTGGCCGAGGACAACATCATCCATGGCCTGGACTTCTGGACCGCCGCCAAGCGTGACTACCTCGAGGCCCTCGACGAGGTGATCCTCAAGCCGCGCGGCCTGGACTACAAGGTCCAGTTCCCTGGACCGACCGGCACCAATGCCGTCGAGGCGGCCATCCGCCTGGCCCGCAACGCCAAGGGCCGCCACAACATCGTCACCTTCACCAACGGCTTCCACGGCGTGACCATGGGGGCGCTGGCCACCACCGGTAACCGCAAGTTCCGCGAGGCCACGGGCGGCGTGCCCACGGTCGGCGGGAGCTTCATGCCCTTCGACGGCTACCTGGGCGAGGGCGCCGACACCCTGGATTACTTCGAGAAGCTGCTCGGCGACAAGTCCGGCGGCCTGGACATCCCGGCGGGGGTGATCGTCGAGACCGTGCAGGGCGAGGGCGGTATCAACGTCGCTGGCCTCGACTGGCTCAAGCGCCTCGAGGGCATCTGCCGCGCCCATGACATCCTGCTGATCGTCGACGACATCCAGGCCGGCTGCGGCCGCACCGGCAAGTTCTTCAGCTTCGAACACGCCGACGTCGTTCCCGATATCGTCACCAACTCCAAGTCGCTCTCCGGCCTCGGCCTGCCGTTCTCCCAGGTGCTGATGCGTCCTGAACTCGATGTCTGGAAGCCGGGCCAGTACAACGGCACCTTCCGCGGCTTCGCGCTTGCCTTCACCACCGCGGCCGCCGCCTTGCGCCACTATTGGAGCGACGACGCCCTGGCCCAGGACGTGGCGCGCAAGGGCGAGGTGGTCGCCAAGCGCTTCCAGAAGATCGCCGGCATGCTCGGCGAACTGGGCATCGAGGCCTCCGAGCGTGGCCGCGGCCTGATGCGCGGGATCGACGTGGGTAGCGGTGACATCGCCGACAAGATCACCCACAAGGCCTTTGAGAACGGGCTGGTCATCGAGACCAGCGGTCAGGACGGCGAGGTAGTCAAGTGCCTCTGCCCGCTGACCATCACCGATGAGGAGCTGGACATGGGCCTCGATATTCTCGAGACCAGCACCAAGCAGGCGCTTAGCTGA

SEQ ID NO.3：ectC (as shown in sequence 3 in the sequence table)

ATGATCGTTCGCAATCTCGATGACGCCCGCAAGACCGACCGCCTGGTCAAGGCCGAAAACGGCAACTGGGACAGCACCCGCCTGAGTCTGGCCGATGATGGCGGCAACTGCTCCTTCCATATCACGCGTATCTACGAAGGCACCGAGACCCACATCCACTACAAGCATCACTTCGAGGCCGTTTTCTGCATCGAAGGCGAGGGCGAGGTGGAAACCCTGGCCGACGGCAAGATCTGGCCGATCAAGCCGGGTGACATCTACATCCTCGACCAGCACGACGAGCACCTGCTGCGCGCCAGCAAGACCATGCACCTGGCCTGCGTGTTCACGCCGGGCCTGACCGGCAACGAGGTGCACCGCGAGGATGGCTCCTACGCGCCGGCCGAGGCCGACGACAAGAAGCCGCTCTGA

SEQ ID No. 4: (as shown in sequence 4 in the sequence table)

MTMNATTEPFTPSADLARPTVADAVVGHEAYPLFIRKPSPDDGWGIYELVKSCPPLDVNSAYAYLLLATQFRDSCAVATNEEGEIVGFVSGYVKSNAPDTYFLWQVAVGEKARGTGLARRLVEAVMTRPEMTEVHHLETTITPDNQASWGLFRRLAERWQAPLNSREYFSTDQLGGEHDPENLVRIGPFQTDRI

SEQ ID No. 5: (as shown in sequence 5 in the sequence table)

MQTQILERMESEVRTYSRSFPVVFTKARNARLTDEDGREYIDFLAGAGTLNYGHNNPHLKQALLDYLAEDGIIHGLDFWTAAKRDYLEALDEVILKPRGLDYKVQFPGPTGTNAVEAAIRLARNAKGRHNIVTFTNGFHGVTMGALATTGNRKFREATGGVPTVGGSFMPFDGYLGEGTDTLDYFEKLLGDKSGGLDIPAGVIVETVQGEGGINVAGLDWLKRLEGICRAHDILLIVDDIQAGCGRTGKFFSFEHADVVPDIVTNSKSLSGLGLPFSQVLMRPELDVWKPGQYNGTFRGFALAFTTAAAALRHYWSDDALAQDVARKGEVVAKRFQKIAGMLGELGIEASERGRGLMRGIDVGSGDIADKITHKAFENGLIIETSGQDGEVVKCLCPLTITDEELDMGLDILETSTKQALS

SEQ ID NO. 6: (as shown in sequence 6 in the sequence table)

MIVRNLDDARKTDRLVKAENGNWDSTRLSLADDGGNCSFHITRIYEGTETHIHYKHHFEAVYCIEGEGEVETLADGKIWPIKPGDIYILDQHDEHLLRASKTMHLACVFTPGLTGNEVHREDGSYAPAEADDKKPL

first, Chassis bacteria gene cluster is extracted from Chassis strainsHalomonas sp. Selecting monoclonal from YL01, culturing in culture solution, and extracting genome;

thirdly, constructing recombinant expression plasmid, and mixing the vector skeleton withectABCConstructing recombinant expression plasmid by gene cluster connection, and connecting the recombinant expression plasmid to a large intestine competent cell for culture;

the fourth step, mutant construction, onectA、ectB、ectCDesigning primers for gene mutation, and constructing mutant plasmids.

The method further comprises:

first, seed solution preparation, construction based on expression vectorsectABCTransferring recombinant expression plasmids of the gene cluster and the mutant thereof into escherichia coli for expression, and culturing the obtained monoclonal to obtain seed liquid;

step two, tetrahydropyrimidine is fermented and cultured, wherein seed liquid is inoculated into a culture medium, and the tetrahydropyrimidine is produced through fermentation and culture;

and thirdly, preparing tetrahydropyrimidine, centrifugally recovering thalli, breaking walls of the thalli, filtering by a membrane to prepare tetrahydropyrimidine supernatant, and detecting and analyzing the product concentration and the like.

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

The following disclosure provides many different embodiments, or examples, for implementing different embodiments of the invention. To simplify the disclosure, specific embodiments or examples are described below. Of course, they are merely examples and are not intended to limit the present invention. In addition, the present invention provides examples of various specific processes and materials, and one of ordinary skill in the art will recognize the applicability of other processes and/or the use of other materials. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, and the like, which are within the capabilities of persons skilled in the art. In addition, unless otherwise indicated, nucleic acids are written from left to right in the 5 'to 3' direction herein.

The invention is described below by way of illustrative specific examples, which do not limit the scope of the invention in any way. Specifically, the following are mentioned: the reagents used in the present invention are commercially available unless otherwise specified.

It is to be noted that technical terms or scientific terms used in the embodiments of the present invention should have the ordinary meanings as understood by those having ordinary skill in the art to which the present disclosure belongs, unless otherwise defined.

Test materials and reagents

1. The strain is as follows: screened by the inventionectABCThe gene cluster is derived from the flora sequencing of the salt lake of Qinghai province. Meanwhile, the method of bacterial strain screening, PCR amplification verification, genome sequencing, fermentation production and the like is used for confirming the source bacterial strain of the gene cluster, and after the bacterial strain identification is carried out on the source bacterial strain, the source bacterial strain is namedHalomonas sp. YL01, biological accession number: GDMCC No. 62420.

2. Culture medium and batch feeding fermentation nutrient components of recombinant escherichia coli JM 109:

(1) LB medium (g/L): 5-20 parts of peptone, 3-10 parts of yeast powder and 10-30 parts of sodium chloride, and adjusting the pH value to 6-10, and using the peptone for culturing recombinant Escherichia coli JM 109; the plate is prepared by adding 1.5-2% agarose.

(2) MM medium (g/L): 10-30 parts of glucose, 0.5-10 parts of urea, 0-10 parts of aspartic acid, 1-20 parts of yeast powder, 0.05-0.6 part of anhydrous magnesium sulfate, 1.5-5.5 parts of monopotassium phosphate, 5-30 parts of sodium chloride, and Fe (III) -NH₄-Citrate 0.05-0.1，CaCl₂·2H₂O 0.02-0.2，ZnSO₄·7H₂O 0.1-0.2，MnCl₂·4H₂O 0.03-0.09，H₃BO₃ 0.3-1，CoCl₂·6H₂O 0.2-0.8， CuSO₄·5H₂O 0.01-0.08，NiCl₂·6H₂O 0.02-0.1，NaMoO₄·2H₂O0.03-0.12, and is used for fermentation of recombinant Escherichia coli JM 109.

(3) Feed I (g/L): glucose 300-1000, urea 20-100, aspartic acid 0-15; feeding II (g/L): glucose 500-1000, urea 2-20.

3. Halomonas sp. YL01 culture medium

(1) 60LB Medium (g/L): peptone 5-20, yeast powder 3-10, sodium chloride 60-80, pH adjusted 8-10 for useHalomonas sp. Sieving and culturing YL 01; the plate is prepared by adding 1.5-2% agarose.

Example 1 tetrahydropyrimidine synthesis gene clusterectABCAcquisition of sequences

Selecting flora of salt lake sludge in Qinghai province, resuspending and diluting the sludge by 10-1000 times by using a sterile culture medium, and performing monoclonal screening on strains by a plate coating method. Screening to obtain different monoclonals, performing PCR amplification verification, and selecting the size and the ratio of the amplified fragmentsectABCThe clone with consistent gene cluster fragments is subjected to sequencing verification analysis, and the tetrahydropyrimidine synthetic gene cluster is obtained after the verification is correctectABCAnd (4) sequencing.

Then, the strain is subjected to whole genome sequencing, the source strain of the gene cluster is confirmed, and the gene cluster is subjected to whole genome sequencingAfter strain identification and genome-wide annotation, the gene was namedHalomonas sp. YL01, biological accession number: GDMCC No. 62420.

Example 2 tetrahydropyrimidine synthesis gene clusterectABCSequence alignment

Analysis by NCBI Blast (https:// Blast. NCBI. nlm. nih. gov/Blast. cgi) toolectABCThe nucleic acid sequence of (1):

ectAand come fromHalomonas aestuarii strain Hb3 (NCBI accession number: CP 018139.1),Halomonas sp. THAF5a (NCBI accession No.: CP045417.1),Halomonas sp. JS92-SW72 (NCBI accession number: CP 032147.1),Halomonas sp. BM-2019(NCBI accession number: CP071922.1),Halomonas elongata (NCBI accession No.: D88359.1)ectAThe sequences have similarity of 94.17%, 93.83%, 91.27%, 90.19% and 88.36%, respectively, and the specific alignment results are shown in FIG. 2;

ectBand come fromHalomonas aestuarii strain Hb3 (NCBI accession No.: CP 018139.1),Halomonas sp. THAF5a (NCBI accession No.: CP045417.1),Halomonas sp. JS92-SW72 (NCBI accession number: CP 032147.1),Halomonas sp. BM-2019(NCBI accession number: CP071922.1),Halomonas elongata (NCBI accession No.: D88359.1)ectBThe specific alignment results are shown in fig. 3 and fig. 4, and have similarities of 91.4%, 91.09%, 88.35%, 87.62% and 85.28%, respectively;

ectCand come fromHalomonas sp. JS92-SW72 (NCBI accession No.: CP 032147.1),Halomonas elongata (NCBI accession No. D88359.1),Halomonas aestuarii strain Hb3 (NCBI accession No.: CP 018139.1),Halomonas sp. THAF5a (NCBI accession No.: CP045417.1),Halomonas sp. BM-2019(NCBI accession number: CP071922.1)ectCThe sequences were 91.99%, 91.71%, 91.3%, 90.7.62% and 88.35% similar, respectively, and the results of the specific alignment are shown in FIG. 5.

Therefore, tetrahydropyrimidine synthesis gene clusterectABCInectAAnd fromHalomonas elongata Of a plurality of halomonas species (NCBI accession No.: D88359.1)ectAAt least 88.36% DNA sequence identitySource type; tetrahydropyrimidine synthesis gene clusterectABCInectBAnd come fromHalomonas elongata Of a plurality of halomonas species (NCBI accession No.: D88359.1)ectBAt least 85.28% DNA sequence homology; tetrahydropyrimidine synthesis gene clusterectABCInectCAnd come fromHalomonas sp. Of a large number of halomonas such as BM-2019(NCBI accession number: CP071922.1)ectCThere is at least 88.35% DNA sequence homology.

Example 3 tetrahydropyrimidine Synthesis Gene ClusterectABCRecombination and mutation of (A)

FIG. 6 shows gene clustersectABCThe recombinant expression map of (1) is as follows:

Halomonas sp. YL01 genome extraction

Will be provided withHalomonas sp. YL01 was inoculated on a 60LB non-resistant plate, inverted cultured at 37 ℃ for 24 hours, and then monocloned was selected to 5 mL of a shake tube of 60LB culture solution, and shake-cultured at 37 ℃ and 180 rpm for 12 hours; extracting 2 mL of bacterial liquid according to bacterial genome DNA extraction kit (purchased from Tiangen Biochemical technology Co., Ltd.)Halomonas sp. YL01 genome.

Expression vector pSEVA321 framework and gene clusterectABCPCR amplification of sequences

According to the expression vector pSEVA321 and gene clusterectABCSequence information, primers were designed using Snapgene software (Version 8.02) with the following sequences:

expression vector-F: TGATAAGCCAGGCATCAAATAAAACG (shown as sequence 7 in the sequence table)

Expression vector-R: CTAGTATTTCTCCTCTTTCTCTAGTATTAAAC (shown as sequence 8 in the sequence table)

ectABCGene cluster-F:

GAGAAAGAGGAGAAATACTAGATGAGTACGCCAATAACACCTTTTACCCC (shown as sequence 9 in the sequence table)

ectABCGene cluster-R: TTTGATGCCTGGCTTATCATTACTCACCCGCGGGTGCTG (shown as sequence 10 in the sequence table)

Vector pSEVA321 and vector obtained in the above 1Halomonas sp.YL01 genomes are respectively used as templates, the total reaction volume is 50 mu L, and the reaction volume is 0.2 mL of PCR tubesThe following ingredients shown in table 1 were added in sequence:

TABLE 1

And (3) after uniform mixing, performing instantaneous centrifugation, wherein the reaction parameters are as follows: denaturation at 98 ℃ for 30 sec; denaturation at 98 ℃ for 10 sec, annealing at 65 ℃ for 30 sec, extension at 72 ℃ for 1.5 min, and final extension at 72 ℃ for 2 min after 35 cycles. The pSEVA321 backbone and gene cluster were recovered using a universal DNA purification kit (available from Tiangen Biochemical technology Ltd.)ectABCThe operation is carried out according to the steps provided by the product specification.

Construction of recombinant expression plasmids

(ii) the pSEVA321 backbone and gene cluster obtained in 2 aboveectABCAnd (3) connecting to construct a recombinant expression plasmid: ligation was performed by T4 DNA Ligase (purchased from New England Biolabs) with a total reaction volume of 20. mu.L, and the following components shown in Table 2 were added sequentially to a 0.2 mL PCR tube:

TABLE 2

After mixing, the mixture was centrifuged instantaneously and ligated overnight at 16 ℃ to obtain a ligation product.

Preparation of competent cells by chemical transformation of Escherichia coli JM109

1) Using LB plate culture medium, using inoculating loop to pick out Escherichia coli (-20 deg.C glycerol preservation strain), grading and streaking on plate, and performing inverted culture at 37 deg.C for 14-16 h;

2) picking activated from LB platesE. coliJM109 single colony is inoculated in 5 mL LB liquid medium, and is subjected to shaking culture at 37 ℃ for 12 h;

3) the above culture was mixed at a ratio of 1: 100 in 100 mL LB liquid medium, 37 ℃ shaking culture until OD600=0.5, placing on ice to stop culture;

4) transferring 1mL of the bacterial liquid into a 1.5 mL centrifuge tube, centrifuging at 4000 rpm and 4 ℃ for 10 min, and removing the supernatant; then, the procedure was carried out according to the instruction of the comparative Cell Preparation Kit (Takara corporation, a Kit for making the large intestine Competent);

5) competent cells were dispensed into 50. mu.L/tube on ice and stored at-80 ℃ to obtain competent cell JM 109.

③ the ligation product transforms the competent cells JM109 of the large intestine

The competent cell JM109 cells obtained from the above ② were thawed in an ice bath immediately after being taken out from a freezer at-80 ℃. Adding the ligation product obtained in the step I into Escherichia coli competent cell JM109, gently mixing, carrying out ice bath for 30 min, carrying out water bath heat shock at 42 ℃ for 90 s, immediately carrying out ice bath for 2 min, adding 0.75 mL of LB liquid culture medium, and recovering at 37 ℃ for 2 h. And (3) taking 100 muL of bacterial liquid, coating the bacterial liquid on an LB (Luria Bertani) flat plate containing Cm resistance (the final concentration is 100 mug/mL), and carrying out inverted culture at 37 ℃ for 12-16 h.

And (4) selecting a positive single colony, inoculating the positive single colony into 5 mL of LB liquid culture medium containing Cm resistance (the final concentration is 100 mug/mL), carrying out overnight culture at 37 ℃ at 180 rpm, verifying the positive single colony through PCR of a bacteria liquid, and carrying out sequencing analysis to show that the recombinant plasmid is successfully constructed.

Construction of mutants

① ectAPrimer design for gene mutation: according to gene clusterectABCAs a result of sequence alignment, N39S and A132T double mutation points were designed, and Snap gene software (Version 8.02) was used to design mutation primers, the primer sequences were as follows (mutated bases are underlined):

39-F：AGCCCCGATGACGGCTGGGGCATCTACG (shown as sequence 11 in the sequence table)

39-R: GGGCTTGCGGATGAACAGCGGATAGGC (shown as sequence 12 in the sequence table)

132-F：ACGGAGGTCCACCACCTCGAGACCACC (shown as sequence 13 in the sequence table)

132-R: CATCTCCGGGCGGGTCATCACGGCTTCCA (shown as sequence 14 in the sequence table)

Extracting the plasmid constructed in the step 3, amplifying by using primers 39-F and 39-R to obtain a linearized plasmid, connecting the linearized plasmid with the plasmid 2, and connecting the linearized plasmid with the plasmid 3 to obtain a mutation plasmid of the amino acid at the position 39; extracting the 39 th amino acid mutation plasmid, and amplifying by using primers 132-F and 132-R to obtain a lineThe plasmid linearization step is the same as the step 2, the linearized plasmid is connected with the step 3 to obtain the 132-position amino acid mutant plasmid, and the 39-position and 132-position amino acid mutant plasmids are the plasmidsectAGene mutation plasmid, amino acid sequence after mutation

② ectBPrimer design for gene mutation: according to gene clusterectABCAs a result of sequence alignment, I59L, N71G, A179T and V381I multi-mutation points were designed, and mutant primers were designed using Snap gene software (Version 8.02), and the primer sequences were as follows (mutated bases are underlined):

59-F：CTAAAGCAGGCGCTGCTCGACTACCT (shown as sequence 15 in the sequence table)

59-R: GTGCGGGTTGTTGTGGCCGTAGTTC (shown as sequence 16 in the sequence table)

71-F：GGAATCATCCATGGCCTGGACTTCTGGA (shown as sequence 17 in the sequence table)

71-R: GTCCTCGGCCAGGTAGTCGAGCAGC (shown as sequence 18 in the sequence table)

179-F：ACCGACACCCTGGATTACTTCGAGAAGCTG (shown as sequence 19 in the sequence table)

179-R: GCCCTCGCCCAGGTAGCCGTCGAAGGGCAT (shown as sequence 20 in the sequence table)

381-F：ATAATCGAGACCAGCGGTCAGGACGG (shown as sequence 21 in the sequence table)

381-R: CAGCCCGTTCTCAAAGGCCTTGTGGGTGATCT (shown as sequence 22 in the sequence table)

The experimental steps for constructing mutant plasmids are the same as those of the experimental stepsectAPrimer design for gene mutation

Extracting the one constructed in the above-mentioned (I)ectAGene mutation plasmid, using primers 59-F and 59-R to amplify to obtain linearized plasmid, the steps are the same as those of 2, then connecting the linearized plasmid, the steps are the same as those of 3, and obtaining 59-position amino acid mutation plasmid; extracting 32-site amino acid mutant plasmid, amplifying by using primers 71-F and 71-R to obtain linearized plasmid, connecting the linearized plasmid with the above step 2, connecting the linearized plasmid with the above step 3 to obtain 71-site amino acid mutant plasmid, constructing 179 and 381-site amino acid mutant plasmids by the same method, and finally obtaining 59, 71, 179 and 381-site amino acid mutant plasmidsectBA gene mutation plasmid.

③ designing primers for the mutation of the ectC gene: according to the alignment result of the gene cluster ectABC sequence, F62Y mutation points are designed, Snap gene software (Version 8.02) is used for designing mutation primers, and the sequences of the primers are as follows (the mutated bases are marked by underlines):

62-F：TACTGCATCGAAGGCGAGGGCGAGGTGGAAAC (shown as sequence 23 in the sequence table)

62-R: GGGCTTGCGGATGAACAGCGGATAGGC (shown as sequence 24 in the sequence table)

Extracting the above-mentionedectBAmplifying the gene mutation plasmid by using primers 62-F and 62-R to obtain a linearized plasmid, connecting the linearized plasmid with the step 2, and connecting the linearized plasmid with the step 3 to obtain the No. 62 amino acid mutation plasmid, wherein the site mutation plasmid is the site mutation plasmidectCA gene mutation plasmid.

EXAMPLE 4 fermentative production of tetrahydropyrimidines

1. Seed liquid preparation

(ii) construction of an expression vector based on pSEVA321ectABCRecombinant expression plasmids of the gene cluster and the mutant thereof are transferred into Escherichia coli JM109 for expression; taking an inoculating loop, streaking the recombinant Escherichia coli JM109 strain on an anti-LB (lysogeny Broth-Marie) plate on a superclean bench, and activating at 37 ℃ for 24 hours until a monoclonal antibody grows out;

selecting the monoclonal antibody in the step 1, inoculating the monoclonal antibody into a shake tube filled with 5 mL of seed culture medium (LB), and culturing for 12 hours at 37 ℃ and 200 rpm;

③ inoculating 200 mu L of the bacterial liquid in the 2 into a 150mL conical flask filled with 20 mL of seed culture medium (LB), and culturing at 37 ℃ and 200 rpm for 12 h;

inoculating 1mL of the bacterial liquid in the 3 into a 500mL conical flask filled with 100 mL of seed culture medium (LB), and culturing at 37 ℃ and 200 rpm for 12 h;

2. fermentative production of tetrahydropyrimidines

I) And (3) shaking flask fermentation: adding 50mL of fermentation medium (MM) into 500mL of the mixture, adjusting the pH value to 7-10 by NaOH, inoculating the seed solution according to the volume ratio of 2.5-5%, controlling the temperature at 35-38 ℃ and the rotating speed at not higher than 220 rpm in the fermentation process, and carrying out fermentation culture for 36-48 h.

II) fermentation in a fermentation tank: adding 3.6L fermentation medium (MM) into 7.5L fermentation tank, adjusting pH to 6-10 with NaOH, inoculating seed liquid at 5-10% volume ratio, controlling temperature at 35-38 deg.C, rotation speed no higher than 1000 rpm, and dissolved oxygen at 5-35% during fermentation. Sampling every 1 h in the fermentation process for OD detection and residual sugar detection:

starting to add the supplementary material I after 6-10 hours from the beginning of fermentation to maintain the glucose concentration at 5-10 g/L;

secondly, feeding II is started 16-20 hours after fermentation is started, and the concentration of glucose is maintained at 5-10 g/L;

③ fermenting for 24-28 hours and then finishing the fermentation.

3. Detection of tetrahydropyrimidines

And (3) breaking the bacterial wall by taking the bacterial strain with a proper dilution factor, centrifuging the bacterial strain at the rotating speed of 12000 rpm for 10 min, taking the supernatant, filtering the supernatant by using a 0.22 mu m microporous filter membrane, and performing HPLC analysis. The liquid phase conditions were as follows: c18 chromatographic column. The mobile phase is acetonitrile (liquid A) and pure water (liquid B), and A: B =70: 30; the sample volume is 10 mu L; the flow rate is 1 mL/min; the detection wavelength is 210 nm, and the tetrahydropyrimidine detected by HPLC is shown in figure 1.

4. Preparation of tetrahydropyrimidines

After the culture is finished, the thalli are recovered by centrifugation, the product of the released intracellular tetrahydropyrimidine is extracted by alcohol, and then the product is concentrated, filtered by a membrane and crystallized to obtain the tetrahydropyrimidine.

Comparative example

Preparing a seed solution:

seed solution preparation and tetrahydropyrimidine fermentation preparation were carried out in the same manner as in example 4, except that the gene clusters used were obtained by the literature methods provided in Table 3.

TABLE 3 comparison of fermentation yields of examples and comparative examples

^aIn the case, the gene cluster of ectoABC before mutation and the TD source of BluepharigenesisComparing the yield of heterologous expression of the ectABC gene cluster, wherein the shake flask fermentation method is the same as that in example 4;

^bin the case, the yield of heterologous expression of the mutant ectABC gene cluster is compared with that of the h.bluePhagenesis TD source ectABC gene cluster, and the shake flask fermentation method is the same;

^cin the case, the yield of heterologous expression of the mutant ectABC gene cluster is compared with that of an ectABC gene cluster from H.bluePhagenesis TD, H.elongata DSM2581, and the fermentation method of a 7-L fermentation tank is adopted.

Ma and the like utilize an ectABC gene cluster derived from a H.bluephasegenetics TD strain to produce tetrahydropyrimidine by using escherichia coli fermentation of genetic engineering, the tetrahydropyrimidine grows for 48 hours in a 500mL shake flask, and the yield of the tetrahydropyrimidine reaches 3.4 g/L; a process for producing tetrahydropyrimidines by fermentation of genetically engineered H.bluegene TD strains, growing 48H in 500mL shake flasks and batch-fed cultivation 28H in 7L bioreactors, with tetrahydropyrimidines contents of 6.3 and 28 g/L (Ma H, ZHao Y, Huang W, et al, Rational flash-tuning of halogenes for co-production of bioplastic PHB and choice [ J ] Nature communications, 2020, 11(1): 1-12; Ning et al, using the H.electron DSM2581 strain-derived ecto ABC gene cluster, by the metabolically engineered E.coli method, tetrahydropyrimidines fermented in shake flasks are not provided, with a yield of up to 25.1g/L in the 7-L fermenter fermentation mode of production of tetrahydropyrimidine and choice, metabolic Engineering, 2016, 36: 10-18.)

As can be seen from the comparison in Table 3, the enzyme catalytic efficiency of the gene cluster coding provided by the invention is better, and after mutation, the efficiency of converting into tetrahydropyrimidine is improved by over 30 percent compared with the traditional method, and the efficiency after mutation is further improved by 20 percent.

All of the above mentioned intellectual property rights are not intended to be restrictive to other forms of implementing the new and/or new products. Those skilled in the art will take advantage of this important information, and the foregoing will be modified to achieve similar performance. However, all modifications or alterations are based on the new products of the invention and belong to the reserved rights.

The foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Sequence listing

<110> Ke Ling carbon Biotech limited in Shenzhen

<120> tetrahydropyrimidine biosynthesis gene cluster, mutant and method for preparing tetrahydropyrimidine

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<212> DNA

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atgacaatga acgcaaccac cgagcccttc acaccctccg ccgacctggc acgccccacc 60

gtggcggacg ccgtggtcgg tcacgaggcc tatccgctgt tcatccgcaa gcccaacccc 120

gatgacggct ggggcatcta cgagctggtc aagtcctgcc ccccgctgga cgtcaactcc 180

gcctatgcct acctgctgct ggcgacccag ttccgcgaca gttgtgccgt ggccaccaac 240

gaggagggcg agatcgtcgg tttcgtctcc ggctacgtga agagcaacgc cccggacacc 300

tacttcctgt ggcaggtggc ggtcggcgag aaggcgcgcg gcaccggcct ggcccggcgc 360

ctggtggaag ccgtgatgac ccgcccggag atggccgagg tccaccacct cgagaccacc 420

atcacccccg acaaccaggc ctcctggggc ctgttccggc ggcttgccga acgctggcag 480

gcgccgctca acagccgcga gtacttctcc accgaccagc tcggtggcga gcacgacccg 540

gaaaacctcg tgcgcatcgg ccccttccag accgatcgca tctga 585

<210> 2

<211> 1266

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<213> Artificial sequences (artificial series)

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atgcagaccc agatcctcga acgcatggag tccgaagttc ggacctattc ccgctccttt 60

ccggtggtct tcaccaaggc ccggaatgcc cgtctgaccg acgaggacgg ccgcgagtac 120

atcgacttcc tggccggtgc cggcaccctg aactacggcc acaacaaccc gcacatcaag 180

caggcgctgc tcgactacct ggccgaggac aacatcatcc atggcctgga cttctggacc 240

gccgccaagc gtgactacct cgaggccctc gacgaggtga tcctcaagcc gcgcggcctg 300

gactacaagg tccagttccc tggaccgacc ggcaccaatg ccgtcgaggc ggccatccgc 360

ctggcccgca acgccaaggg ccgccacaac atcgtcacct tcaccaacgg cttccacggc 420

gtgaccatgg gggcgctggc caccaccggt aaccgcaagt tccgcgaggc cacgggcggc 480

gtgcccacgg tcggcgggag cttcatgccc ttcgacggct acctgggcga gggcgccgac 540

accctggatt acttcgagaa gctgctcggc gacaagtccg gcggcctgga catcccggcg 600

ggggtgatcg tcgagaccgt gcagggcgag ggcggtatca acgtcgctgg cctcgactgg 660

ctcaagcgcc tcgagggcat ctgccgcgcc catgacatcc tgctgatcgt cgacgacatc 720

caggccggct gcggccgcac cggcaagttc ttcagcttcg aacacgccga cgtcgttccc 780

gatatcgtca ccaactccaa gtcgctctcc ggcctcggcc tgccgttctc ccaggtgctg 840

atgcgtcctg aactcgatgt ctggaagccg ggccagtaca acggcacctt ccgcggcttc 900

gcgcttgcct tcaccaccgc ggccgccgcc ttgcgccact attggagcga cgacgccctg 960

gcccaggacg tggcgcgcaa gggcgaggtg gtcgccaagc gcttccagaa gatcgccggc 1020

atgctcggcg aactgggcat cgaggcctcc gagcgtggcc gcggcctgat gcgcgggatc 1080

gacgtgggta gcggtgacat cgccgacaag atcacccaca aggcctttga gaacgggctg 1140

gtcatcgaga ccagcggtca ggacggcgag gtagtcaagt gcctctgccc gctgaccatc 1200

accgatgagg agctggacat gggcctcgat attctcgaga ccagcaccaa gcaggcgctt 1260

agctga 1266

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atcacgcgta tctacgaagg caccgagacc cacatccact acaagcatca cttcgaggcc 180

gttttctgca tcgaaggcga gggcgaggtg gaaaccctgg ccgacggcaa gatctggccg 240

atcaagccgg gtgacatcta catcctcgac cagcacgacg agcacctgct gcgcgccagc 300

aagaccatgc acctggcctg cgtgttcacg ccgggcctga ccggcaacga ggtgcaccgc 360

gaggatggct cctacgcgcc ggccgaggcc gacgacaaga agccgctctg a 411

<210> 4

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<213> Artificial sequences (artificial series)

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Met Thr Met Asn Ala Thr Thr Glu Pro Phe Thr Pro Ser Ala Asp Leu

1 5 10 15

Ala Arg Pro Thr Val Ala Asp Ala Val Val Gly His Glu Ala Tyr Pro

20 25 30

Leu Phe Ile Arg Lys Pro Ser Pro Asp Asp Gly Trp Gly Ile Tyr Glu

35 40 45

Leu Val Lys Ser Cys Pro Pro Leu Asp Val Asn Ser Ala Tyr Ala Tyr

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Leu Leu Leu Ala Thr Gln Phe Arg Asp Ser Cys Ala Val Ala Thr Asn

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Glu Glu Gly Glu Ile Val Gly Phe Val Ser Gly Tyr Val Lys Ser Asn

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Ala Pro Asp Thr Tyr Phe Leu Trp Gln Val Ala Val Gly Glu Lys Ala

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Arg Gly Thr Gly Leu Ala Arg Arg Leu Val Glu Ala Val Met Thr Arg

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Pro Glu Met Thr Glu Val His His Leu Glu Thr Thr Ile Thr Pro Asp

130 135 140

Asn Gln Ala Ser Trp Gly Leu Phe Arg Arg Leu Ala Glu Arg Trp Gln

145 150 155 160

Ala Pro Leu Asn Ser Arg Glu Tyr Phe Ser Thr Asp Gln Leu Gly Gly

165 170 175

Glu His Asp Pro Glu Asn Leu Val Arg Ile Gly Pro Phe Gln Thr Asp

180 185 190

Arg Ile

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<213> Artificial sequences (artificial series)

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Met Gln Thr Gln Ile Leu Glu Arg Met Glu Ser Glu Val Arg Thr Tyr

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Ser Arg Ser Phe Pro Val Val Phe Thr Lys Ala Arg Asn Ala Arg Leu

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Thr Asp Glu Asp Gly Arg Glu Tyr Ile Asp Phe Leu Ala Gly Ala Gly

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Thr Leu Asn Tyr Gly His Asn Asn Pro His Leu Lys Gln Ala Leu Leu

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Asp Tyr Leu Ala Glu Asp Gly Ile Ile His Gly Leu Asp Phe Trp Thr

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Ala Ala Lys Arg Asp Tyr Leu Glu Ala Leu Asp Glu Val Ile Leu Lys

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Pro Arg Gly Leu Asp Tyr Lys Val Gln Phe Pro Gly Pro Thr Gly Thr

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Asn Ala Val Glu Ala Ala Ile Arg Leu Ala Arg Asn Ala Lys Gly Arg

115 120 125

His Asn Ile Val Thr Phe Thr Asn Gly Phe His Gly Val Thr Met Gly

130 135 140

Ala Leu Ala Thr Thr Gly Asn Arg Lys Phe Arg Glu Ala Thr Gly Gly

145 150 155 160

Val Pro Thr Val Gly Gly Ser Phe Met Pro Phe Asp Gly Tyr Leu Gly

165 170 175

Glu Gly Thr Asp Thr Leu Asp Tyr Phe Glu Lys Leu Leu Gly Asp Lys

180 185 190

Ser Gly Gly Leu Asp Ile Pro Ala Gly Val Ile Val Glu Thr Val Gln

195 200 205

Gly Glu Gly Gly Ile Asn Val Ala Gly Leu Asp Trp Leu Lys Arg Leu

210 215 220

Glu Gly Ile Cys Arg Ala His Asp Ile Leu Leu Ile Val Asp Asp Ile

225 230 235 240

Gln Ala Gly Cys Gly Arg Thr Gly Lys Phe Phe Ser Phe Glu His Ala

245 250 255

Asp Val Val Pro Asp Ile Val Thr Asn Ser Lys Ser Leu Ser Gly Leu

260 265 270

Gly Leu Pro Phe Ser Gln Val Leu Met Arg Pro Glu Leu Asp Val Trp

275 280 285

Lys Pro Gly Gln Tyr Asn Gly Thr Phe Arg Gly Phe Ala Leu Ala Phe

290 295 300

Thr Thr Ala Ala Ala Ala Leu Arg His Tyr Trp Ser Asp Asp Ala Leu

305 310 315 320

Ala Gln Asp Val Ala Arg Lys Gly Glu Val Val Ala Lys Arg Phe Gln

325 330 335

Lys Ile Ala Gly Met Leu Gly Glu Leu Gly Ile Glu Ala Ser Glu Arg

340 345 350

Gly Arg Gly Leu Met Arg Gly Ile Asp Val Gly Ser Gly Asp Ile Ala

355 360 365

Asp Lys Ile Thr His Lys Ala Phe Glu Asn Gly Leu Ile Ile Glu Thr

370 375 380

Ser Gly Gln Asp Gly Glu Val Val Lys Cys Leu Cys Pro Leu Thr Ile

385 390 395 400

Thr Asp Glu Glu Leu Asp Met Gly Leu Asp Ile Leu Glu Thr Ser Thr

405 410 415

Lys Gln Ala Leu Ser

420

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Met Ile Val Arg Asn Leu Asp Asp Ala Arg Lys Thr Asp Arg Leu Val

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Lys Ala Glu Asn Gly Asn Trp Asp Ser Thr Arg Leu Ser Leu Ala Asp

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Asp Gly Gly Asn Cys Ser Phe His Ile Thr Arg Ile Tyr Glu Gly Thr

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Glu Thr His Ile His Tyr Lys His His Phe Glu Ala Val Tyr Cys Ile

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Glu Gly Glu Gly Glu Val Glu Thr Leu Ala Asp Gly Lys Ile Trp Pro

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Ile Lys Pro Gly Asp Ile Tyr Ile Leu Asp Gln His Asp Glu His Leu

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Leu Arg Ala Ser Lys Thr Met His Leu Ala Cys Val Phe Thr Pro Gly

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Leu Thr Gly Asn Glu Val His Arg Glu Asp Gly Ser Tyr Ala Pro Ala

115 120 125

Glu Ala Asp Asp Lys Lys Pro Leu

130 135

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<213> Artificial sequences (artificial series)

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tgataagcca ggcatcaaat aaaacg 26

<210> 8

<211> 32

<212> DNA

<213> Artificial sequences (artificial series)

<400> 8

ctagtatttc tcctctttct ctagtattaa ac 32

<210> 9

<211> 50

<212> DNA

<213> Artificial sequences (artificial series)

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gagaaagagg agaaatacta gatgagtacg ccaataacac cttttacccc 50

<210> 10

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<212> DNA

<213> Artificial sequences (artificial series)

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tttgatgcct ggcttatcat tactcacccg cgggtgctg 39

<210> 11

<211> 28

<212> DNA

<213> Artificial sequences (artificial series)

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agccccgatg acggctgggg catctacg 28

<210> 12

<211> 27

<212> DNA

<213> Artificial sequences (artificial series)

<400> 12

gggcttgcgg atgaacagcg gataggc 27

<210> 13

<211> 27

<212> DNA

<213> Artificial sequences (artificial series)

<400> 13

acggaggtcc accacctcga gaccacc 27

<210> 14

<211> 29

<212> DNA

<213> Artificial sequences (artificial series)

<400> 14

catctccggg cgggtcatca cggcttcca 29

<210> 15

<211> 26

<212> DNA

<213> Artificial sequences (artificial series)

<400> 15

ctaaagcagg cgctgctcga ctacct 26

<210> 16

<211> 25

<212> DNA

<213> Artificial sequences (artificial series)

<400> 16

gtgcgggttg ttgtggccgt agttc 25

<210> 17

<211> 28

<212> DNA

<213> Artificial sequences (artificial series)

<400> 17

ggaatcatcc atggcctgga cttctgga 28

<210> 18

<211> 25

<212> DNA

<213> Artificial sequences (artificial series)

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gtcctcggcc aggtagtcga gcagc 25

<210> 19

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<212> DNA

<213> Artificial sequences (artificial series)

<400> 19

accgacaccc tggattactt cgagaagctg 30

<210> 20

<211> 30

<212> DNA

<213> Artificial sequences (artificial series)

<400> 20

gccctcgccc aggtagccgt cgaagggcat 30

<210> 21

<211> 26

<212> DNA

<213> Artificial sequences (artificial series)

<400> 21

ataatcgaga ccagcggtca ggacgg 26

<210> 22

<211> 32

<212> DNA

<213> Artificial sequences (artificial series)

<400> 22

cagcccgttc tcaaaggcct tgtgggtgat ct 32

<210> 23

<211> 32

<212> DNA

<213> Artificial sequences (artificial series)

<400> 23

tactgcatcg aaggcgaggg cgaggtggaa ac 32

<210> 24

<211> 27

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<213> Artificial sequences (artificial series)

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gggcttgcgg atgaacagcg gataggc 27

Claims

1. A tetrahydropyrimidine biosynthesis gene cluster, comprising: at least comprises 3 genes which are respectively:

ectAthe gene has a nucleic acid sequence shown as SEQ ID NO. 1;

ectBthe gene and the nucleic acid sequence are shown as SEQ ID NO. 2;

ectCthe gene and the nucleic acid sequence are shown as SEQ ID NO. 3.

2. The tetrahydropyrimidine biosynthesis gene cluster according to claim 1 wherein: the gene cluster is derived from halomonas strainHalomonas sp. YL01, deposited at the Guangdong province culture Collection on 24/4 2022 with the deposit number GDMCC No. 62420.

3. The method for obtaining the tetrahydropyrimidine biosynthesis gene cluster according to claim 1 or 2, comprising:

secondly, PCR amplification verification;

and thirdly, sequencing the genome to obtain a gene cluster.

4. A tetrahydropyrimidine biosynthetic gene cluster forming mutant according to claim 1 or 2 comprising:

the nucleic acid sequence is shown as SEQ ID NO.1ectAThe gene has the following mutations on the basis of the original sequence: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is at the 39 th amino acidThe acid is replaced by N to S, the 132 th amino acid is replaced by A to T, and the amino acid sequence of the mutated polypeptide is shown as SEQ ID NO. 4.

5. The tetrahydropyrimidine biosynthesis gene cluster forming mutant according to claim 1 or 2 comprising:

the nucleic acid sequence is shown as SEQ ID NO.2ectBThe gene has the following mutations on the basis of the original sequence: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is changed from I to L at the 59 th amino acid, from N to G at the 71 th amino acid, from A to T at the 179 th amino acid and from V to I at the 381 th amino acid, and the amino acid sequence of the polypeptide after mutation is shown as SEQ ID NO. 5.

6. A tetrahydropyrimidine biosynthetic gene cluster forming mutant according to claim 1 or 2 comprising:

the nucleic acid sequence is shown as SEQ ID NO.3ectCThe gene has the following mutations on the basis of the original sequence: the amino acid sequence of the polypeptide corresponding to the nucleic acid sequence is replaced by Y from F at the 62 nd amino acid, and the amino acid sequence of the mutated polypeptide is shown as SEQ ID NO. 6.

7. A method for constructing a mutant according to any one of claims 4 to 6, which comprises:

firstly, extracting a chassis bacteria gene cluster;

thirdly, constructing recombinant expression plasmids;

and fourthly, constructing a mutant.

8. An expression vector or a recombinant microorganism comprising the synthetic gene cluster of claim 1 or 2 or the mutant of any one of claims 4 to 6.

9. Use of the gene cluster of claim 1 or 2, or the mutant of any one of claims 4 to 6, or the expression vector or the recombinant microorganism engineering bacteria of claim 8 in the synthesis of tetrahydropyrimidine.

10. The method for producing tetrahydropyrimidine by using the recombinant microorganism engineering bacteria of claim 8, wherein tetrahydropyrimidine is produced by converting glucose into whole cells using the recombinant microorganism engineering bacteria.