CN115851510A - Halomonas and application thereof in co-production of tetrahydropyrimidine and polyhydroxyalkanoates - Google Patents

Abstract

The invention provides a halomonas strainHalomonasYL01, by using the strain as the Chassis bacteria, adopting a molecular means to over-express tetrahydropyrimidine synthetic gene cluster and introducing an exogenous PHB synthetic approach, the coproduction of tetrahydropyrimidine can be realizedPyridine and polyhydroxyalkanoates, and greatly improves the yield of the two.

Description

Halomonas and application thereof in co-production of tetrahydropyrimidine and polyhydroxyalkanoates

Technical Field

The invention relates to the technical field of synthetic biology, in particular to halomonas and application thereof in co-production of tetrahydropyrimidine and polyhydroxyalkanoate.

Background

Tetrahydropyrimidine is a cyclic amino acid derivative, having hydrophilic and zwitterionic characteristics, and is one of the typical microbial compatible solutes. It can not only balance the osmotic pressure inside and outside the cell as osmotic pressure compensation solute, but also help the protease, nucleic acid, biological membrane in the cell and even the whole cell resist the harmful effect of denaturant such as high temperature, freezing, drying, oxygen free radical radiation, urea, etc., and can also assist the protein to assemble and fold correctly in the cell, and plays the role of molecular chaperone. Because the tetrahydropyrimidine has strong biomacromolecule (exopolysaccharide, enzyme, DNA, antibody and the like) and cell stabilizing effect, and can protect cells from being influenced by extreme environments such as high osmotic pressure, high temperature and the like, the tetrahydropyrimidine has good application potential in the fields of fine chemical engineering, environmental management, agricultural biotechnology, biomedicine and the like.

The presence of naturally synthesized tetrahydropyrimidine metabolic pathways within halomonas was first reported. Through decades of researches, the synthetic pathway of tetrahydropyrimidine has been deeply developed at the gene level, the enzyme level and the regulation level. In all the anabolic pathways of tetrahydropyrimidine, aspartate semialdehyde (L-aspartate-B-semialdehyde) in the lysine biosynthetic pathway is taken as a precursor, and the operon of the linked ectABC gene cluster which is highly conserved in evolution is relied on. Structural genes ectB, ectA and ectC respectively code L-diaminobutyric acid transaminase (EctB), L-diaminobutyric acid acetyltransferase (EctA) and tetrahydropyrimidine synthase (EctC), and tetrahydropyrimidine is synthesized through 3 steps of catalysis.

The inventor carries out macro genome sequencing analysis after salt lake sludge sampling, and obtains the ectABC gene cluster with a brand-new sequence through sequence comparison and screening, compared with the traditional ectABC gene cluster operon, the ectoABC gene cluster operon can realize one-step catalytic synthesis of tetrahydropyrimidine, but has a large rising space in the aspect of yield.

On the other hand, polyhydroxyalkanoates (PHA) are intracellular polyesters synthesized by many bacteria, exist mainly as storage substances of carbon sources and energy sources in living bodies, and have many excellent properties such as physicochemical properties similar to those of synthetic plastics and biodegradability, biocompatibility, optical activity, piezoelectricity, gas barrier properties, and the like, which synthetic plastics do not have. The polyhydroxyalkanoate has wide application prospect in biodegradable packaging materials, tissue engineering materials, slow release materials, electrical materials and medical materials.

The inventors have found that Halomonas sp.YL01 has its own PHA biosynthetic pathway, mainly synthesized via the acetyl-CoA pathway, which is catalyzed by two molecules of acetyl-CoA, in turn via β -ketothiolase (phaA), NADPH-dependent acetyl-CoA reductase (phaB) and PHB synthase (phaC), to form poly-3-hydroxybutyrate (PHB). However, despite the above-mentioned metabolic pathways for PHA production, the yields are low, limiting its industrial application.

The inventor further researches and discovers that in the Halomonas sp.YL01, the ectocytic transport effect of the tetrahydropyrimidine is good and can reach more than 60 percent, and because of the advantage, the coproduction of the tetrahydropyrimidine and the polyhydroxy fatty acid ester can be completely considered. Therefore, the inventor considers that the Halomonas sp.YL01 is directly adopted as the chassis bacteria to co-produce tetrahydropyrimidine and polyhydroxy fatty acid ester, and the yield of the tetrahydropyrimidine and the polyhydroxy fatty acid ester is improved by over-expressing a tetrahydropyrimidine synthesis gene cluster and introducing an exogenous PHB synthesis way, so that the large-scale industrialization of the tetrahydropyrimidine and the polyhydroxy fatty acid ester is better realized.

Disclosure of Invention

The invention aims to solve the technical problem of providing a Halomonas sp.YL01 strain, which is taken as a basidiomycete, adopts a molecular means to over-express a tetrahydropyrimidine synthetic gene cluster and introduces an exogenous PHB synthetic approach, can realize the co-production of tetrahydropyrimidine and polyhydroxy fatty acid ester and greatly improve the yield of the tetrahydropyrimidine and the polyhydroxy fatty acid ester.

Based on the above, the invention provides an application of Halomonas sp.YL01 in co-production of tetrahydropyrimidine and polyhydroxyalkanoate.

The Halomonas sp.YL01 strain is deposited at the institute of microbiology, academy of sciences of Guangdong province (center for microbiological analysis and detection of Guangdong province), and has a date of 24/4/2022 and a deposit number of GDMCC No. 62420.

The Halomonas sp.YL01 strain comprises a gene cluster ectABC which at least comprises 3 genes which are respectively:

the nucleic acid sequence of the ecto-A gene is shown as SEQ ID NO. 1;

the nucleic acid sequence of the gene ectB is shown as SEQ ID NO. 2;

the nucleotide sequence of the gene ecto-C is shown in SEQ ID NO. 3.

The invention provides a Halomonas sp.YL01-1 strain, which is obtained by over-expressing an ectABC gene cluster on the Halomonas sp.YL01 strain.

The invention also provides a preparation method of the Halomonas sp.YL01-1, which comprises the following steps:

firstly, constructing an ectABC suicide plasmid pRE112-ectABC carrying a gene cluster;

secondly, transforming the suicide plasmid pRE112-ectABC into Escherichia coli S17-1;

and thirdly, combining the strain to an underpan bacterium Halomonas sp.YL01 to obtain a strain with the ectABC gene cluster over-expressed, wherein the strain is named Halomonas sp.YL01-1.

The invention provides a Halomonas sp.YL01-2 strain, which is obtained by adopting PCT540 gene heterologous expression on a Halomonas sp.YL01-1 strain of a Halomonas underpinning.

The invention provides a preparation method of Halomonas sp.YL01-2, which comprises the following steps:

firstly, constructing a suicide plasmid pRE112-PCT 540;

secondly, transforming the suicide plasmid pRE112-PCT540 into Escherichia coli S17-1;

and thirdly, combining to the Halomonas sp.YL01-1 to obtain the Halomonas sp.YL01 strain of PCT540 gene for heterologous expression and designation of Halomonas sp.YL01-2.

The invention also provides a method for co-producing tetrahydropyrimidine and polyhydroxyalkanoate, which comprises the following steps:

firstly, preparing seed liquid of Halomonas sp.YL01-2;

step two, preparing fermentation liquor;

and step three, fermentation culture and coproduction of tetrahydropyrimidine and polyhydroxyalkanoate.

The invention has the following beneficial technical effects: the Halomonas sp.YL01 is provided, and by taking the Halomonas sp.YL01 as a basidiomycete, and adopting a molecular means to over-express a tetrahydropyrimidine synthesis gene cluster and introducing an exogenous PHB synthesis way, the coproduction of tetrahydropyrimidine and polyhydroxy fatty acid ester can be realized, and the yield of the tetrahydropyrimidine and polyhydroxy fatty acid ester can be greatly improved.

Drawings

FIG. 1 is a diagram of a halomonas pcr containing the ectABC gene cluster obtained from a salt lake sludge sample in Qinghai province in example 1;

FIG. 2 is a diagram showing a recombinant expression map of gene cluster ectABC;

FIG. 3 is a schematic diagram of the induction of tetrahydropyrimidine production by E.coli;

FIG. 4 is a schematic representation of the suicide plasmid map of gene cluster ectABC;

FIG. 5 is a map of the suicide plasmid of gene pct 540.

Biological material preservation information

YL01, classified and named Halomonas sp.YL01, deposited at the institute of microbiology, guangdong province, institute of microbiology, analysis and detection, with strain number GDMCC.No62420, date of deposit 2022, 24 months 04, and deposit address of institute of microbiology, guangdong province (analysis and detection center for microorganisms, guangdong province)

Detailed Description

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. Further, while the present invention provides examples of various specific processes and materials, 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.

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. Culture medium of recombinant escherichia coli DH5 alpha and batch feed fermentation nutrient components

(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, wherein the peptone is used for culturing recombinant Escherichia coli JM 109; the plate is prepared by adding 1.5-2% agarose.

(2) MM medium (g/L): 10 to 30 portions of glucose, 0.5 to 10 portions of urea, 0 to 10 portions of aspartic acid, 1 to 20 portions of yeast powder, 0.05 to 0.6 portion of anhydrous magnesium sulfate, 1.5 to 5.5 portions of monopotassium phosphate, 5 to 30 portions of sodium chloride, fe (III) -NH ₄ -Citrate 0.05-0.1，CaCl ₂ ·2H ₂ O0.02-0.2，ZnSO ₄ ·7H ₂ O 0.1-0.2，MnCl ₂ ·4H ₂ O 0.03-0.09，H ₃ BO ₃ 0.3-1，CoCl ₂ ·6H ₂ O 0.2-0.8，CuSO ₄ ·5H ₂ O 0.01-0.08，NiCl ₂ ·6H ₂ O 0.02-0.1，NaMoO ₄ ·2H ₂ O0.03-0.12, and is used for fermentation of recombinant Escherichia coli JM 109.

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

2. Culture medium of Halomonas sp.YL01

(1) 60LB medium (g/L): 5-20 parts of peptone, 3-10 parts of yeast powder, 60-80 parts of sodium chloride, and 8-10 parts of pH regulation, and the peptone is used for screening and culturing Halomonas sp.YL01; the plate is prepared by adding 1.5-2% agarose.

Example 1 acquisition of Halomonas sp.YL01

Taking 1g of salt lake sludge sample in Qinghai province, continuously diluting with sterile water, coating on an LB plate containing NaCl (60 g/L), culturing at 37 ℃ for 48h, picking out a single colony after the colony grows out, continuously performing streak subculture for 30d, and performing acclimatization and screening until pure bacteria are obtained. The obtained pure bacterium 16S rDNA is amplified, sequenced and compared with the sequence to obtain the Halomonas sp.YL01 of the Halomonas of the invention, and the biological preservation number is as follows: no62420 with gdmcc. The genome was then sent to Kyowa Biometrics, guangzhou for sequencing analysis to obtain an ectABC gene cluster, as shown in FIG. 1.

The ectABC gene cluster comprises

The nucleic acid sequence of the ecto-A gene is shown as SEQ ID NO. 1;

the nucleic acid sequence of the ectoB gene is shown as SEQ ID NO. 2;

the nucleic acid sequence of the ecto-C gene is shown in SEQ ID NO. 3.

SEQ ID NO.1：ectA

ATGACAATGAACGCAACCACCGAGCCCTTCACACCCTCCGCCGACCTGGCACGCCCCACCGTGGCGGACGCCGTGGTCGGTCACGAGGCCTATCCGCTGTTCATCCGCAAGCCCAACCCCGATGACGGCTGGGGCATCTACGAGCTGGTCAAGTCCTGCCCCCCGCTGGACGTCAACTCCGCCTATGCCTACCTGCTGCTGGCGACCCAGTTCCGCGACAGTTGTGCCGTGGCCACCAACGAGGAGGGCGAGATCGTCGGTTTCGTCTCCGGCTACGTGAAGAGCAACGCCCCGGACACCTACTTCCTGTGGCAGGTGGCGGTCGGCGAGAAGGCGCGCGGCACCGGCCTGGCCCGGCGCCTGGTGGAAGCCGTGATGACCCGCCCGGAGATGGCCGAGGTCCACCACCTCGAGACCACCATCACCCCCGACAACCAGGCCTCCTGGGGCCTGTTCCGGCGGCTTGCCGAACGCTGGCAGGCGCCGCTCAACAGCCGCGAGTACTTCTCCACCGACCAGCTCGGTGGCGAGCACGACCCGGAAAACCTCGTGCGCATCGGCCCCTTCCAGACCGATCGCATCTGA

SEQ ID NO.2：ectB

ATGCAGACCCAGATCCTCGAACGCATGGAGTCCGAAGTTCGGACCTATTCCCGCTCCTTTCCGGTGGTCTTCACCAAGGCCCGGAATGCCCGTCTGACCGACGAGGACGGCCGCGAGTACATCGACTTCCTGGCCGGTGCCGGCACCCTGAACTACGGCCACAACAACCCGCACATCAAGCAGGCGCTGCTCGACTACCTGGCCGAGGACAACATCATCCATGGCCTGGACTTCTGGACCGCCGCCAAGCGTGACTACCTCGAGGCCCTCGACGAGGTGATCCTCAAGCCGCGCGGCCTGGACTACAAGGTCCAGTTCCCTGGACCGACCGGCACCAATGCCGTCGAGGCGGCCATCCGCCTGGCCCGCAACGCCAAGGGCCGCCACAACATCGTCACCTTCACCAACGGCTTCCACGGCGTGACCATGGGGGCGCTGGCCACCACCGGTAACCGCAAGTTCCGCGAGGCCACGGGCGGCGTGCCCACGGTCGGCGGGAGCTTCATGCCCTTCGACGGCTACCTGGGCGAGGGCGCCGACACCCTGGATTACTTCGAGAAGCTGCTCGGCGACAAGTCCGGCGGCCTGGACATCCCGGCGGGGGTGATCGTCGAGACCGTGCAGGGCGAGGGCGGTATCAACGTCGCTGGCCTCGACTGGCTCAAGCGCCTCGAGGGCATCTGCCGCGCCCATGACATCCTGCTGATCGTCGACGACATCCAGGCCGGCTGCGGCCGCACCGGCAAGTTCTTCAGCTTCGAACACGCCGACGTCGTTCCCGATATCGTCACCAACTCCAAGTCGCTCTCCGGCCTCGGCCTGCCGTTCTCCCAGGTGCTGATGCGTCCTGAACTCGATGTCTGGAAGCCGGGCCAGTACAACGGCACCTTCCGCGGCTTCGCGCTTGCCTTCACCACCGCGGCCGCCGCCTTGCGCCACTATTGGAGCGACGACGCCCTGGCCCAGGACGTGGCGCGCAAGGGCGAGGTGGTCGCCAAGCGCTTCCAGAAGATCGCCGGCATGCTCGGCGAACTGGGCATCGAGGCCTCCGAGCGTGGCCGCGGCCTGATGCGCGGGATCGACGTGGGTAGCGGTGACATCGCCGACAAGATCACCCACAAGGCCTTTGAGAACGGGCTGGTCATCGAGACCAGCGGTCAGGACGGCGAGGTAGTCAAGTGCCTCTGCCCGCTGACCATCACCGATGAGGAGCTGGACATGGGCCTCGATATTCTCGAGACCAGCACCAAGCAGGCGCTTAGCTGA

SEQ ID NO.3：ectC

ATGATCGTTCGCAATCTCGATGACGCCCGCAAGACCGACCGCCTGGTCAAGGCCGAAAACGGCAACTGGGACAGCACCCGCCTGAGTCTGGCCGATGATGGCGGCAACTGCTCCTTCCATATCACGCGTATCTACGAAGGCACCGAGACCCACATCCACTACAAGCATCACTTCGAGGCCGTTTTCTGCATCGAAGGCGAGGGCGAGGTGGAAACCCTGGCCGACGGCAAGATCTGGCCGATCAAGCCGGGTGACATCTACATCCTCGACCAGCACGACGAGCACCTGCTGCGCGCCAGCAAGACCATGCACCTGGCCTGCGTGTTCACGCCGGGCCTGACCGGCAACGAGGTGCACCGCGAGGATGGCTCCTACGCGCCGGCCGAGGCCGACGACAAGAAGCCGCTCTGA

Example 2 recombinant expression of Gene Cluster ectABC

FIG. 2 shows a schematic diagram of the recombinant expression map of gene cluster ectABC, which is as follows:

1. halomonas sp.YL01 genome extraction

Inoculating Halomonas sp.YL01 on a 60LB non-resistant plate, performing inverted culture at 37 ℃ for 24h, selecting a monoclonal to a shake culture tube of 5mL 60LB culture solution, and performing shaking culture at 37 ℃ and 180rpm for 12h; 2mL of the bacterial solution was taken, and Halomonas sp.YL01 genome was extracted according to a bacterial genome DNA extraction kit (purchased from Tiangen Biochemical technology Co., ltd.).

2. PCR amplification of expression vector pSEVA321 framework and gene cluster ectABC sequence

According to the sequence information of the expression vector pSEVA321 and the gene cluster ectABC, primers are designed by utilizing Snapgene software (Version 8.02), and the sequences of the primers are as follows:

expression vector-F: cccgcgggtg agtaatgata ctagtagcgg ccgctc

Expression vector-R: tattggcgta ctcatctagt atttcccctc tttctctagt attaaacaaa attatttgt

ectABC gene cluster-F: gaggggaaat actagatgag tacgccaata acacctttta ccc

ectABC gene cluster-R: ccgctactag tatcattact cacccgcggg tgc

Using the vector pSEVA321 and the Halomonas sp.YL01 genome obtained in 1 as templates, the total reaction volume was 50. Mu.L, and the following components shown in Table 1 were sequentially added to a 0.2mL PCR tube:

TABLE 1

And (3) instantly centrifuging after uniformly mixing, wherein the reaction parameters are as follows: denaturation at 98 ℃ for 30sec; denaturation at 98 ℃ for 10sec, annealing at 65 ℃ for 30sec, extension at 72 ℃ for 1.5min, and final extension at 72 ℃ for 2min after 35 cycles. The pSEVA321 backbone and gene cluster ectABC were recovered using a universal DNA purification kit (available from Tiangen Biochemical technology Ltd.) following the procedures provided in the product instructions.

3. Construction of recombinant expression plasmids

(1) Connecting the pSEVA321 skeleton obtained in the step 2 and the gene cluster ectABC to construct a recombinant expression plasmid: ligation was performed by T4DNA Ligase (from New England Biolabs) in a total reaction volume of 20. Mu.L, and the following components shown in Table 2 were sequentially added to a 0.2mL PCR tube:

TABLE 2

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

(2) Preparation of competent cells for chemical transformation of Escherichia coli DH5 alpha

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 14-16h;

2) Picking activated E.coli DH5 alpha single colonies from an LB plate, inoculating the single colonies into 5mL of LB liquid culture medium, and carrying out shake culture at 37 ℃ for 12h;

3) The above culture was mixed at a ratio of 1:100 in 100mL LB liquid medium, and shake-culturing at 37 ℃ to OD ₆₀₀ =0.5 or so, and placed on ice to stop culturing;

4) Transferring 1mL of the above bacterial liquid into a 1.5mL centrifuge tube, centrifuging at 4000rpm at 4 ℃ for 10min, 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 split into 50. Mu.L/tube on ice and stored at-80 ℃ to obtain competent cell DH 5. Alpha.

(3) Ligation product transformed into large intestine competent cell DH5 alpha

The competent cell DH 5. Alpha. Of (2) above was taken out from a freezer at-80 ℃ and thawed in a quick ice bath. And (2) adding the ligation product obtained in the step (1) into an escherichia coli competent cell DH5 alpha, gently mixing, carrying out ice bath for 30min, carrying out water bath heat shock at 42 ℃ for 90s, immediately carrying out ice bath for 2min, adding 0.75mL of LB liquid culture medium, and recovering for 2h at 37 ℃. 100 μ L of the bacterial suspension was spread on an LB plate containing Cm resistance (final concentration: 100 μ g/mL) and cultured in an inverted state at 37 ℃ for 12 to 16 hours.

And (3) selecting a positive single colony, inoculating the positive single colony into 5mL LB liquid culture medium containing Cm resistance (the final concentration is 100 mu g/mL), culturing at 37 ℃ overnight at 180rpm, verifying the positive single colony through bacterial liquid PCR (polymerase chain reaction) and indicating that the recombinant plasmid is successfully constructed through sequencing analysis.

EXAMPLE 3 fermentative production of tetrahydropyrimidines

1. Seed liquid preparation

(1) Taking an inoculating loop, streaking the recombinant Escherichia coli DH5 alpha strain onto an LB (LB) plate (Cm) on a superclean bench, activating for 24 hours at 37 ℃, and growing a single clone;

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

(3) 200. Mu.L of the above-mentioned bacterial suspension 2 was inoculated into a 150mL Erlenmeyer flask containing 20mL of seed medium (LB), and cultured at 37 ℃ and 200rpm for 12 hours.

2. Shaking flask fermentation production of tetrahydropyrimidine

Adding 50mL LB,50 μ L chloramphenicol into 500mL conical flask, adjusting pH to 7-10 with NaOH, inoculating seed liquid at 2.5-5% volume ratio, controlling temperature at 35-38 deg.C and rotation speed no higher than 220rpm during fermentation, and changing inducer concentration (10 rpm) in fermentation system ^-4 -10 ^-2 M), fermenting and culturing for 48h, and measuring OD, tetrahydropyrimidine and dry weight (DCM) after the fermentation is finished.

As can be seen from FIG. 3, the gene cluster ectABC can play a role in tetrahydropyrimidine synthesis, and the promoter can normally express the ectABC as shown by adding different concentrations of inducer arabinose into a fermentation system, and when the concentration of the inducer is 10 ^-3 M, the growth of the whole thallus, the content of tetrahydropyrimidine and the dry weight of the thallus show the optimal level.

Example 4 overexpression of Gene Cluster ectABC to achieve yield increase of tetrahydropyrimidine

1. Construction of ectoABC suicide plasmid carrying Gene cluster (pRE 112-ectoABC)

PCR amplification of expression vector pRE112 backbone and gene cluster ectABC sequence

Expression vector-F: ggcaccggac gctaaggtga tatagagtgt atcgcgcaaa

Expression vector-R: gcggtgtgga ggcatgatag tctcgaatct tccgaccaat ga

ectABC gene cluster-F:

agattcgaga ctatcatgcc tccacaccgc tcgtcacatc ctgttgcgtt cactggaatc ccagtataaa gt

ectABC gene cluster-R: ccgctactag tatcattact cacccgcggg tgc

The procedure for constructing the plasmid was the same as in example 2.

2. Suicide plasmid pRE112-ectABC was transformed into E.coli S17-1 and ligated into Halomonas sp. YL01

(1) The plasmid constructed in this example 1 was extracted with pRE112-ectABC using a plasmid Mini kit (purchased from Tiangen Biochemical technology Co., ltd.) according to the procedures provided in the product instructions, and the extracted plasmid was stored at-20 ℃ until transformation for use.

(2) Preparation of chemically transformed competent cells of Escherichia coli S17-1 (same as in example 2)

(3) Ligation products transformed competent cells S17-1 in the Large intestine (same as example 2)

And (3) selecting a positive single colony, inoculating the positive single colony into 5mL LB liquid culture medium containing Cm resistance (the final concentration is 100 mu g/mL), culturing at 37 ℃ overnight at 180rpm, verifying the positive single colony through bacterial liquid PCR (polymerase chain reaction) and indicating that the recombinant plasmid is successfully constructed through sequencing analysis.

(4) Joining

Donor bacteria (S17-1) and acceptor bacteria (Halomonas sp. YL01) are respectively cultured in LB and 60LB liquid culture media with corresponding resistance overnight at 37 ℃ for 12-14h, are respectively diluted according to the volume ratio of 1. Dripping 200 μ L of nonreactive 60LB liquid culture medium on the lawn, spreading the lawn, placing the flat plate upside down in a 37 deg.C incubator, culturing for 48h, and growing single clone.

(5) Homologous recombination

And (4) continuously carrying out streak subculture on the single clone in the step (4) for one week to obtain a strain with over-expressed ectABC gene cluster, and naming the strain as Halomonas sp.

Respectively fermenting the constructed strains Halomonas sp.YL01-1 and Halomonas sp.YL01 in 500ml shake flask and 6L fermentation tank system, and comparing the contents of produced tetrahydropyrimidine and PHB

Example 5 fermentative production of tetrahydropyrimidine and PHB by overexpression of the Strain Halomonas sp.YL01-1 and the Strain Halomonas sp.YL01

(1) Preparation of seed liquid of Halomonas sp.YL01 shake flask

(1) Strain activation

Taking strains in a refrigerator at the temperature of 80 ℃ below zero in a laboratory, selecting a bacterium solution by using a gun head, streaking and inoculating the strain on a flat solid culture medium (5 g/L of yeast powder, 10g/L of tryptone, 60g/L of sodium chloride and 8.5 of pH), and culturing for 24 hours at the temperature of 37 ℃.

(2) First-stage seed culture:

a single colony was picked and inoculated into a 12ml shake tube (5 ml 60LB medium: yeast powder 5g/L; tryptone 10g/L; sodium chloride 60g/L; pH 8.5), and the culture was incubated for 12 hours at 220rpm on a shaker 37 ℃.

(3) Secondary seed culture:

the first-order bacterial liquid (1-2% inoculum size) was aspirated, inoculated into a 150ml conical flask (20ml 60LB medium), and cultured on a shaker at 37 ℃ and 220rpm for 12h.

(2) Preparation of seed liquid of Halomonas sp.YL01-1 shake flask

(1) Bacterial activation

Taking strains in a refrigerator at the temperature of 80 ℃ below zero in a laboratory, selecting a bacterium solution by using a gun head, streaking and inoculating the strain on a flat solid culture medium (5 g/L of yeast powder, 10g/L of tryptone, 60g/L of sodium chloride, 8.5 of pH and 1 thousandth of chloramphenicol), and culturing for 24 hours at the temperature of 37 ℃.

(2) First-order seed culture:

a single colony is selected and inoculated in a 12ml shake tube (5 ml 60LB culture medium: 5g/L yeast powder; 10g/L tryptone; 60g/L sodium chloride; pH 8.5; 1 ‰ chloramphenicol), and the culture solution is cultured for 12h at 220rpm in a shaking table at 37 ℃.

(3) Secondary seed culture:

the first-order bacterial liquid (1-2% inoculum size) was taken, inoculated into a 150ml conical flask (20ml 60LB medium; chloramphenicol 1 ‰), and cultured in a shaker at 37 deg.C and 220rpm for 12h.

(3) Preparation of seed liquid of Halomonas sp.YL01 fermentation tank

(1) Strain activation and first-order seed liquid culture are as described above (1)

(2) Secondary seed liquid culture:

the first-order bacterial liquid (1-2% inoculum size) is absorbed and inoculated into a 500ml conical flask (100ml 60LB culture medium), and the mixture is cultured for 12 hours at the temperature of 37 ℃ and the speed of 220rpm on a shaking table.

(4) Preparation of seed liquid of Halomonas sp.YL01-1 fermentation tank

(1) Activation of strains and first-order seed liquid culture (2)

(2) Secondary seed liquid culture:

the first-order bacterial liquid (1-2% inoculum size) is absorbed and inoculated in a 500ml conical flask (100ml 60LB culture medium; chloramphenicol 1 ‰), and then cultured for 12h at 220rpm on a shaker at 37 ℃.

(5) Preparation of fermentation broth

A component I: mgSO (MgSO) ₄ 10g/L；CO(NH ₂ ) ₂ 30g/L；

And (2) component II: KH (Perkin Elmer) ₂ PO ₄ 175g/L

Component III is prepared by mixing 5g/L Fe (III) -NH ₄ -Citrate and 2g/L CaCl ₂ ·2H ₂ Taking 100ml of O; taking 10ml of solution (containing ZnSO) ₄ ·7H ₂ O 0.1g/L；MnCl ₂ ·4H ₂ O 0.03g/L；H ₃ BO ₃ 0.3g/L；CoCl ₂ ·6H ₂ O 0.2g/L；CuSO ₄ ·5H ₂ O

0.01g/L；NiCl ₂ ·6H ₂ O 0.02g/L；NaMoO ₄ ·2H ₂ O0.03 g/L) and 90 is addedmixing with ml deionized water, and adjusting pH to 4.5-5.5 with 5M NaOH

Bottom materials: 50g/L of sodium chloride; yeast extract 1g/L

Carbon source: glucose 30g/L

(6) Fermentation culture

Inoculating the seed liquid at 2.5-5% into 500ml conical flask (45 ml of base material; 1ml of component I; 1ml of component II; 1ml of component III; and 1ml of glucose, adjusting pH to 8.5-9.5 with 5M NaOH), and culturing at 220rpm for 48h at 37 ℃ on a shaking table.

Inoculating the seed solution 2.5-5% into 6L fermentation tank (total volume of culture medium is 3.6L), and fermenting for 40-48h.

Note: when the strain Halomonas sp. YL01-1 containing plasmid is fermented, 1 per thousand of chloramphenicol needs to be added.

(7) Extraction and determination of tetrahydropyrimidine products

Dry cell weight (CDW) by placing 15ml fermented bacterial liquid in 50ml centrifuge tube, centrifuging at 8000rpm at room temperature for 6min, and removing supernatant; washing twice with deionized water; freeze-drying for 15 hours by a freeze dryer; and (5) weighing.

Extracting tetrahydropyrimidine product by taking 5ml of residual bacteria liquid after fermentation into a 50ml centrifuge tube (and adding ddH ₂ Diluting the bacterial liquid by 10 times by using O or deionized water), crushing cells by using a high-pressure cell crusher, and centrifuging at 12000rpm for 10min at room temperature; the supernatant was filtered through a 0.22 μm filter and stored in a liquid chromatography sample vial.

And (3) determining the content of tetrahydropyrimidine: using High Performance Liquid Chromatography (HPLC) with C18 column; the mobile phase is acetonitrile (liquid A) and pure water (liquid B), and A: B = 70; the sample volume is 10 mu L; the flow rate is 1mL/min; the detection wavelength is 210nm. Detection by HPLC.

(8) PHB extraction and content determination

Dry cell weight (CDW) by placing 15ml fermented bacteria liquid in 50ml centrifuge tube, centrifuging at 8000rpm at room temperature for 6min, and removing supernatant; washing with deionized water twice; freeze-drying for 15 hours by a freeze dryer; and (5) weighing.

Determining the content of PHB: then, 2ml of an esterification solution (containing methanol, 3% (v/v) concentrated sulfuric acid (98%, w/w) and 1g/L benzoic acid) and 2ml of chloroform were added to 40mg of the lyophilized cells, and esterified at 100 ℃ for about 4 hours. PHB standard 40mg was treated as reference.

After methanolysis, the samples were tested for PHB content on a GC-2014 gas chromatograph (Shimadzu, japan). The initial temperature is maintained at 80 ℃ for 1.5min;

in the first stage, the temperature is increased to 140 ℃ at a rate of 30 ℃/min;

in the second stage, the temperature is increased to 240 ℃ at a speed of 40 ℃/min, and the process takes 2min;

the total analysis time was 8min;

the injection temperature was 240 ℃ and the detector temperature was 250 ℃

(9) Results of fermentation

The measurements of the different product contents were carried out by means of different instruments and the results are given in table 3.

TABLE 3 determination of tetrahydropyrimidine and PHB content under different fermentation systems

As can be seen from the results in Table 3, the content of tetrahydropyrimidine produced by the strain Halomonas sp.YL01-1 which overexpresses the gene cluster ectABC is remarkably increased, the dry weight of cells is also improved to a certain extent, but the production capacity of PHB is to be improved.

Example 6 overexpression of the PCT540 Gene in the Halomonas sp. YL01-1 Strain

PCT540 is allenyl-CoA transferase from the strain Clostridium propionicum (PCT carries out a gene mutation at V193A and four nucleotide mutations T78C, T669C, A1125G, and T1158C) (Choi et al, 2016). It can specifically obtain 3HB-coA, and greatly increase the content of produced PHB.

Construction of suicide plasmid pRE112-PCT540

PCR amplification of expression vector pRE112 backbone and PCT540 sequence

Expression vector-F:

gctggcattg aacacatgcc tccacaccgc tcgtcacatc ctgttgcgtt cactggaatc ccagtatagc at

expression vector-R: ctacgcgagc agtaacccct aactcccccc tg

PCT540-F：atgagaaagg ttcccattat taccgcagat gaggctg

PCT540--R：tcaggacttc atttccttca gacccattaa gccttctgca

The procedure for constructing the plasmid was the same as in example 2.

The suicide plasmid pRE112-PCT540 was transformed into E.coli S17-1 and ligated to Halomonas sp.YL01-1 (same as example 3) to obtain a Halomonas sp.YL01 strain with PCT540 gene heterologously expressed, which was named Halomonas sp.YL01-2.

Example 7Halomonas sp.YL01-1, halomonas sp.YL01-2 and Halomonas sp.YL01 fermentations were compared

(1) Preparation of seed liquid

The same as in example 5.

(2) Preparation of fermentation broth

A component I: mgSO (MgSO) ₄ 10g/L；CO(NH ₂ ) ₂ 30g/L；

And (2) component II: KH (Perkin Elmer) ₂ PO ₄ 175g/L

And (3) component III: adding 5g/L Fe (III) -NH ₄ -Citrate and 2g/L CaCl ₂ ·2H ₂ Taking 100ml of O; taking 10ml of solution (containing ZnSO) ₄ ·7H ₂ O 0.1g/L；MnCl ₂ ·4H ₂ O 0.03g/L；H ₃ BO ₃ 0.3 g/L；CoCl ₂ ·6H ₂ O 0.2g/L；CuSO ₄ ·5H ₂ O

0.01g/L；NiCl ₂ ·6H ₂ O 0.02g/L；NaMoO ₄ ·2H ₂ O0.03 g/L) and 90ml of deionized water are added and mixed, and finally the pH value is adjusted to 4.5-5.5 by 5M NaOH

Bottom materials: 50g/L of sodium chloride; yeast extract 1g/L

Carbon source: glucose 50g/L

(3) Fermentation culture

Inoculating the seed liquid into a 500ml conical flask (bottom material 45ml; component I1ml; component II 1ml; component III 1ml; glucose 3ml, adjusting pH to 8.5-9.5 with NaOH of 5M; and culturing the strain containing plasmid at 37 deg.C and 220rpm for 48h in a shaking table.

(4) Extraction and determination of tetrahydropyrimidine and PHB

The same procedure as in example 5 was followed.

(5) Results of fermentation

Comparison of the tetrahydropyrimidines and the PHB content produced by the different strains under the same culture conditions is given in Table 2.

TABLE 2 fermentation results of different strains

The PHB content is increased remarkably by introducing PCT540 gene, and the yield of basic tetrahydropyrimidine is not reduced obviously.

Example 8 comparison of Halomonas sp. YL01-2 with fermentation of other Halomonas

Through the same technical scheme of the case, the strains which are used for co-producing tetrahydropyrimidine and PHB and are obtained by carrying out molecular modification on Halonoas sp.DSM2581, halomonas sp.TD01 and Halomonas sp.B01 with different chassis in the same way as in the embodiment 4 and the embodiment 6 respectively have the codes of Halonoas sp.DSM2581-2, halomonas sp.TD01-2 and Halomonas sp.B01-2. The tetrahydropyrimidine and PHB contents produced by the different strains, using the same culture conditions of example 7, are given in Table 3.

TABLE 3 fermentation results of different strains

Through the modification of different chassis, the effect of the Halomonas sp.YL01-2 co-production of tetrahydropyrimidine and PHB is better, and the dry cell weight, the tetrahydropyrimidine content and the PHB content are all at the upstream level.

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.

Claims (9)

1. Halomonas strainHalomonas Application of YL01 in co-production of tetrahydropyrimidine and polyhydroxyalkanoate.

2. Use of halomonas as claimed in claim 1 wherein: the halomonas strainHalomonas YL01, deposited at the institute of microbiology, academy of sciences of Guangdong province (Guangdong province center for microbiological analysis and detection), with a date of 24/4/2022, and a deposit number of GDMCC No62420.

3. Use of halomonas as claimed in claim 1 or 2 wherein: the halomonas strainHalomonas YL01 comprises a gene clusterectABCIt 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.

4. Halomonas strainHalomonas YL01-1, characterized in that: by the halomonas strain of claim 1Halomonas Overexpression on sp.YL01ectABCAnd (5) obtaining a gene cluster.

5. The halomonas strain of claim 4Halomonas A method for preparing sp.yl01-1, characterized in that: the method comprises the following steps:

in a first step, a carrierReason clusterectABCSuicide plasmidpRE112-ectABCConstructing;

second step, suicide plasmidpRE112-ectABCTransforming to Escherichia coli S17-1;

third step, conjugation to Chassis bacteriaHalomonas YL01, obtainingectABCThe strain with the gene cluster over-expressed is namedHalomonas sp. YL01-1。

6. Halomonas strainHalomonas YL01-2, characterized in that: the strain Halomonas underpinna as set forth in claim 4Halomonas On sp.YL01-1PCT540The gene is obtained by heterologous expression.

7. Halomonas strainHalomonas A method for preparing sp.yl01-2, characterized in that: the method comprises the following steps:

first, suicide plasmidpRE112-PCT540Constructing;

second, suicide plasmidpRE112-PCT540Transforming to Escherichia coli S17-1;

third step, the process as claimed in claim 4Halomonas YL01-1, obtainingPCT540Expressed by heterologous expression of genesHalomonas YL01-1 Strain, namedHalomonas sp.YL01-2。

8. A method for co-producing tetrahydropyrimidine and polyhydroxyalkanoate, comprising:

in the first step, the halomonas strain of claim 6Halomonas Preparing an sp.YL01-2 seed solution;

secondly, preparing fermentation liquor;

and step three, fermentation culture and coproduction of tetrahydropyrimidine and polyhydroxyalkanoate.

9. Use of the microorganism engineering bacteria of any one of claims 1, 4 and 6 in the synthesis of tetrahydropyrimidine and/or polyhydroxyalkanoates.

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