CN112961815B

CN112961815B - Genetic engineering bacterium for high yield of tetrahydropyrimidine and application thereof

Info

Publication number: CN112961815B
Application number: CN202110219102.2A
Authority: CN
Inventors: 李莎; 张皓; 徐虹; 王鑫沂; 王瑞; 黄巍巍
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2021-02-26
Filing date: 2021-02-26
Publication date: 2022-07-01
Anticipated expiration: 2041-02-26
Also published as: CN112961815A

Abstract

The invention discloses a genetically engineered bacterium for high yield of tetrahydropyrimidine, which is E.coli ETC15, and a host cell of the genetically engineered bacterium is E.coli MG1655, and comprises the following components: two genes, lysA and pykF, are defective; ectoine synthesis gene cluster ectABC gene controlled by trc promoter; an inulin exonuclease cscA gene controlled by the trc promoter; ask (M68V) derived from Streptomyces albus under the control of trc promoter and fruK, gapA, ppc derived from Escherichia coli under the control of trc promoter. The genetic engineering bacteria can efficiently synthesize high-added-value product tetrahydropyrimidine by using the cheap non-grain raw material jerusalem artichoke crude extract, and effectively improve the yield of tetrahydropyrimidine while reducing the production cost.

Description

Genetic engineering bacterium for high yield of tetrahydropyrimidine and application thereof

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a genetic engineering bacterium for high yield of tetrahydropyrimidine and application thereof.

Background

Tetrahydropyrimidine (Ectoine), also known as 1, 4, 5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid, is an important amino acid derivative, is a main substance for halophilic and salt-tolerant microorganisms to adapt to osmotic stress, can slow down the poison of high temperature, freeze thawing, drought, radiation, chemical reagents and the like to protein, nucleic acid, biomembrane and whole cells, and can be used as a macromolecular protective agent, a cell repairing agent and a whitening agent to have wide application prospect in the fields of biology, medicine, cosmetics and the like. The chemical structural formula is as follows:

with the expanding application range of tetrahydropyrimidine, the market demand is increasing, and the production of tetrahydropyrimidine on the market is mainly obtained by high-density fermentation of halophilic microorganisms, particularly halophilic monads. This process is produced by using a method called "bacterial milking" in which cells are cultured under high osmotic pressure and tetrahydropyrimidine is accumulated inside the cells, then tetrahydropyrimidine is stimulated to be released from the inside to the outside of the cells by hypotonic shocking, and then the hypertonic culturing and hypotonic shocking are repeated to obtain a higher concentration of tetrahydropyrimidine. This process is complicated and requires high salt conditions for the cultivation, and therefore requires high equipment, resulting in a very expensive production. At present, some progress is made on the research of engineering bacteria for producing tetrahydropyrimidine. CN201810996222.1 discloses a recombinant Escherichia coli and application of synthesizing ectoine, wherein the recombinant Escherichia coli is obtained by knocking out a diaminopimelate decarboxylase lysA gene of Escherichia coli E.coli MG1655 and introducing ectoine synthesis gene cluster ectABC. The thalli after the induction expression of the invention takes L-sodium aspartate as a substrate to prepare tetrahydropyrimidine through biotransformation. After 20-30h of conversion, the yield of the tetrahydropyrimidine reaches 2.5-3.5g/L, and the method has good industrial application value. CN201710012845.6 discloses a genetically engineered bacterium for producing tetrahydropyrimidine by xylose induction, a construction method and application thereof. The strain heterologously expresses an ectABC gene cluster from halomonas elongata on an escherichia coli chromosome, reconstructs a tetrahydropyrimidine synthesis path, constructs a plasmid-free system, induces RNA polymerase from T7 bacteriophage by utilizing a xylose promoter, and simultaneously starts target genes to efficiently express by combining with a T7 strong promoter system, so that the yield of tetrahydropyrimidine reaches 12-16g/L after 20-28h of shake flask fermentation, and the yield of tetrahydropyrimidine reaches 35-50g/L after 24-40h of 5L fermentation tank fermentation. However, the prior art has the problems of low utilization rate of carbon sources, high production cost and the like because the carbon sources are mostly from grains such as glucose and the like, so that the development of a strain capable of producing tetrahydropyrimidine by using cheap non-grain raw materials has important significance.

With the rapid development of the fermentation industry, the method presents a vigorous demand for industrial grains, and the cost of raw materials is still an important factor for restricting the large-scale production of the fermentation industry. The jerusalem artichoke is a sustainable and low-cost non-grain biological manufacturing raw material, has important application value in the field of biochemical engineering, and mainly relates to multiple fields of biological energy, platform chemicals, medicines, foods and the like. But compared with the starch industry, the deep processing, development and application of the starch are still insufficient, and the starch has great development and utilization potential and space.

Disclosure of Invention

The purpose of the invention is as follows: in order to solve the problems of low yield, low yield and high production cost in the process of producing tetrahydropyrimidine by using genetically engineered bacteria, the invention constructs genetically engineered escherichia coli, so that the high-added-value product tetrahydropyrimidine can be efficiently synthesized by using cheap non-grain raw material jerusalem artichoke crude extract, and the yield of tetrahydropyrimidine is effectively improved while the production cost is reduced.

In order to achieve the above object, the present invention provides a genetically engineered bacterium for high yield of tetrahydropyrimidine, wherein the genetically engineered bacterium is e.coli ETC15, and a host cell of the genetically engineered bacterium is e.coli MG1655, comprising: two genes, lysA and pykF, are defective; ecto-ABC gene of ectoine synthesis gene cluster controlled by trc promoter; an inulin exonuclease cscA gene controlled by the trc promoter; ask (M68V) derived from Streptomyces albus under the control of trc promoter; the trc promoter controls fruK, gapA, ppc from E.coli.

Preferably, the ectoABC gene of the ectoine synthetic gene cluster is derived from any one of Halomonas meibomiae, Halobacterium halodurans, Halomonas elongata or Cladosporium; the inulase cscA gene is derived from bacillus amyloliquefaciens. In the prior report, high-activity inulinase mainly comes from some fungi, and few inulinase expressed by extracellular direct secretion exists in bacteria, while CscA can be directly expressed by extracellular secretion to hydrolyze inulin, and Escherichia coli is given the capability of synthesizing tetrahydropyrimidine by fermentation with crude extract of Jerusalem artichoke (or inulin) as a fermentation carbon source.

Preferably, the nucleotide sequence of the encoding gene lysA is shown as a sequence table SEQ ID NO 1;

the nucleotide sequence of the coding gene pykF is shown in a sequence table SEQ ID NO. 2;

the nucleotide sequence of the coding gene ectABC gene is shown in a sequence table SEQ ID NO. 3;

the nucleotide sequence of the coding gene cscA is shown in a sequence table SEQ ID NO. 4;

the nucleotide sequence of the coding gene ask (M68V) is shown in a sequence table SEQ ID NO. 5;

the nucleotide sequence of the coding gene fruK is shown in a sequence table SEQ ID NO. 6;

the nucleotide sequence of the coding gene gapA is shown as a sequence table SEQ ID NO. 7;

the nucleotide sequence of the coding gene ppc is shown as a sequence table SEQ ID NO. 8.

The invention further provides a construction method of the genetic engineering bacteria, which comprises the following steps:

(1) cloning a tetrahydropyrimidine synthesis gene cluster ectABC and an inulin exonuclease gene cscA by taking a pTrc99a plasmid as a vector to obtain a recombinant plasmid named as pTETCsA, wherein the two genes are controlled by independent trc promoters, and the recombinant plasmid is transformed into Escherichia coli E.coli MG1655 to construct an initial strain capable of producing tetrahydropyrimidine by using the jerusalem artichoke crude extract, and the initial strain is named as E.coli ETCO;

(2) knocking out lysA and pykF which encode diaminopimelate decarboxylase and pyruvate kinase in E.coli ETCO, and accumulating oxaloacetate synthesis precursor phosphoenolpyruvate, named E.coli ETOK, while weakening the metabolic flux from L-aspartate-beta-semialdehyde to lysine;

(3) the recombinant plasmid pBT-GPAF is obtained by taking pBAD33 plasmid as a carrier to serially express fruK, gapA and ppc derived from escherichia coli and ask (M68V) derived from streptomyces albus, the four genes are controlled by independent trc promoters, a fructose phosphorylation pathway and a glycolysis pathway are enhanced, sufficient oxaloacetate and L-aspartic acid-beta-semialdehyde which are precursors for synthesizing aspartic acid and tetrahydropyrimidine are provided, the recombinant plasmid is transformed into escherichia coli E.coli ETOK, and a strain which finally utilizes the jerusalem artichoke crude extract to produce tetrahydropyrimidine with high yield is constructed and named E.coli ETC 15.

Preferably, the ectopyrimidine synthesis gene cluster ectABC gene is derived from Halomonas meibomiae, the inulinase CsA gene is derived from Bacillus amyloliquefaciens, and the preservation number of the Bacillus amyloliquefaciens is CCTCC No: M2016346.

The invention further provides application of the genetic engineering bacteria in preparation of tetrahydropyrimidine.

Specifically, the steps for preparing tetrahydropyrimidine by shake flask fermentation are as follows:

(1) seed culture: activating the genetic engineering bacteria for producing tetrahydropyrimidine by a plate, inoculating the activated genetic engineering bacteria to a baffle shake flask with a seed culture medium, wherein the inoculation amount is 1-10%, and carrying out shake culture at 30-40 ℃ and 220rpm for 8-12 h;

(2) fermentation culture: inoculating the seed culture obtained in the step (1) to a 500ml baffle shake flask filled with 30-80ml of fermentation medium by an inoculation amount of 5% -15%, performing fermentation culture at 34-40 ℃ and 220rpm for 36-48h in a fermentation period, and finally obtaining 15-20g/L tetrahydropyrimidine.

Wherein the fermentation medium in the step (2) comprises the following components: 5-20g/L of organic nitrogen source, 50-150g/L of carbon source, 5-20g/L of inorganic nitrogen source, 5-10g/L of glycerol, 2-8g/L of monopotassium phosphate, 0.5-1g/L of magnesium sulfate heptahydrate, 0.5-1g/L of sodium chloride, 0.1g/L of ampicillin sodium, 0.025g/L of chloramphenicol and 5-20g/L, IPTG 0.1.1-1 mM of basic magnesium carbonate.

The method for preparing tetrahydropyrimidine by fermentation in a fermentation tank comprises the following steps:

(1) seed culture: activating the genetic engineering bacteria producing tetrahydropyrimidine by a plate, inoculating the activated genetic engineering bacteria to a primary seed culture medium, culturing for 10-12h at 30-37 ℃ and 200rpm, inoculating the activated genetic engineering bacteria to a secondary seed culture medium by 1-10% of inoculum size, and culturing for 8-12h at 37 ℃ and 180-220 rpm;

(2) transferring the seed culture obtained in the step (1) to a fermentation tank filled with a fermentation medium according to the inoculation amount of 1-10% for fermentation culture at 34-40 ℃ for 36-48h, and finally fermenting with the crude extract of the jerusalem artichoke to obtain 45-60g/L tetrahydropyrimidine.

Wherein, the growth is controlled by two different feeding modes in the fermentation culture process, in the growth stage, pH feedback feeding is adopted to reduce the generation of acetic acid, feeding is started at pH6.5-7.5, alkali liquor is not fed in the stage, and the dissolved oxygen is controlled at 40-50%; and (3) entering a product synthesis stage after the target biomass is quickly reached, wherein fed-batch materials are adopted in the product synthesis stage, the concentration of residual sugar is controlled to be 1-5g/L, the pH value is adjusted to be 7.0-8.0 by 20-30% ammonia water, the dissolved oxygen is controlled to be 10% -20%, and sufficient carbon and nitrogen sources are ensured to supply to prolong the product synthesis period.

Preferably, the components of the fermentation medium include: 5-20g/L of organic nitrogen source, 1-20g/L of carbon source, 5-20g/L of inorganic nitrogen source, 5-10g/L of glycerol, 2-4g/L of monopotassium phosphate, 0.5-1g/L of magnesium sulfate heptahydrate, 0.05-0.1g/L of trisodium citrate, 0.05-0.1g/L of manganese sulfate monohydrate, 0.05-0.1g/L of ferrous sulfate heptahydrate, 0.1g/L of ampicillin sodium, and 0.025g/L, IPTG 0.1.1-1 mM of chloramphenicol.

Preferably, the carbon source includes, but is not limited to: one or more of inulin, Jerusalem artichoke crude extract, fructose, and glucose, preferably inulin, Jerusalem artichoke crude extract.

Organic nitrogen sources include, but are not limited to: one or more of peptone, yeast powder, beef extract, soybean meal, corn steep liquor and urea, preferably yeast powder. Inorganic nitrogen sources include, but are not limited to: one or more of ammonium sulfate, ammonium chloride, ammonium nitrate and sodium nitrate, preferably ammonium sulfate.

Has the advantages that: the invention introduces a tetrahydropyrimidine synthesis way into the escherichia coli, and simultaneously introduces a novel secreted inulin exonuclease, so that the escherichia coli can produce tetrahydropyrimidine by using a non-grain raw material jerusalem artichoke crude extract which cannot be utilized as a carbon source. And the capability of the engineering strain for synthesizing tetrahydropyrimidine by using the crude extract of the jerusalem artichoke is further improved by coexpressing key genes of a fructose phosphorylation pathway (fruK), a glycolysis pathway (gapA) and a precursor substance synthesis pathway (ppc, ask (M68V)) and knocking out a metabolic side pathway (lysA) and a byproduct pathway (pykF). Compared with other patents, the yield is greatly improved, the yield of the tetrahydropyrimidine can reach 15-20g/L after the final shake flask fermentation is carried out for 36-48h, the yield of the tetrahydropyrimidine can reach 45-60g/L after the fermentation is carried out for 36-48h, and the used raw materials are cheaper and have development and utilization significance and potential.

Drawings

FIG. 1 is a diagram of metabolic engineering of a strain of the invention;

FIG. 2 is a fermentation graph of genetically engineered Escherichia coli ETC15 fermenter for preparing tetrahydropyrimidine.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The examples will help to understand the present invention given the detailed embodiments and the specific operation procedures, but the scope of the present invention is not limited to the examples described below.

The material sources used in the construction process of the genetic engineering bacteria for producing tetrahydropyrimidine by using the jerusalem artichoke crude extract are as follows:

all primers and DNA sequences were synthesized by Kinry Biotechnology Ltd;

sequencing all plasmids and DNA fragments by the Kinry Biotechnology Limited company;

extracting plasmids by using a plasmid small-amount DNA extraction kit (AP-MN-P-250G) provided by Axygen company;

the recovery of the DNA fragment is carried out by using a DNA gel recovery kit (AP-MN-P-250G) provided by Axygen company;

extracting bacterial genome by using a bacterial genome DNA extraction Kit (TIANAmp Bacteria DNA Kit) provided by Tiangen Biochemical technology Limited;

the DNA fragment PCR is performed by selecting Kodaq 2X PCR MasterMix with dye high fidelity enzyme of Aibimeng biotechnology limited;

the ligase used for One-Step Cloning and connection is selected from ligase (Clonexpress II One Step Cloning Kit) of Novozan Biotechnology GmbH;

the restriction enzyme is restriction enzyme from Baojizishi science and technology Limited.

Gene knockout and plasmid construction in the following examples were verified by PCR and then verified by sequencing by general biosystems (Anhui) Inc.

Example 1 genetic engineering initial strain e.coli ETCO was constructed using a crude extract of jerusalem artichoke to produce tetrahydropyrimidine.

(1) The fragment of ectopyrimidine synthesis gene cluster ectABC (NCBI accession No. MW414529) was made to have homology arms at both ends for homologous recombination with the vector fragment. The plasmid pTrc99a was digested simultaneously with Kpn I and BamH I and the product was purified. The gene fragment of ectABC and the linearized vector are connected by a homologous recombination kit (Novozam, C112-1) to obtain a plasmid pTrc-ectABC.

(2) Taking the genome of bacillus amyloliquefaciens (CCTCC No: M2016346) as a template, taking SEQ ID NO:11 and SEQ ID NO:12 as primers to carry out PCR amplification to obtain a gene fragment cscA, purifying a PCR product, taking plasmid pTrc99a as a template, taking SEQ ID NO:13 and SEQ ID NO:14 as primers to carry out PCR amplification to obtain a promoter gene fragment Trc, and purifying the PCR product. And (3) performing overlap extension PCR amplification by using the purified Trc and cscA as templates and SEQ ID NO 13 and SEQ ID NO 12 as primers to obtain a gene fragment Trc-cscA and purifying a product. Plasmid pTrc-ectABC was digested simultaneously with BamH I and Sal I and the product was purified. The purified linearized plasmid and the gene fragment Trc-cscA are connected by a homologous recombination kit (Novozam, C112-1) to obtain a plasmid pTETCSCA.

(3) Transforming the plasmid obtained in step 2) into e.coli MG1655 to obtain e.coli ETCO.

Example 2 construction of genetically engineered Strain E

(1) Knockout of lysA Gene

1) Knocking out the genes by adopting Red homologous recombination technology. And carrying out PCR amplification by taking pKD3 as a template and SEQ ID NO 15 and SEQ ID NO 16 as primers to obtain a lysA gene knockout fragment, wherein the gene knockout fragment consists of a lysA gene upstream homology arm gene fragment, a chloramphenicol resistance gene fragment and a downstream homology arm gene fragment.

2) And (3) performing electric shock transformation on the gene knockout fragment into E.coli ETCO containing pKD46 plasmid to obtain a positive transformant, and transferring the positive transformant into pCP20 plasmid to eliminate chloramphenicol resistance genes in the positive transformant to obtain lysA gene knockout bacterium E.coli ETCO.

(2) Knock-out of pykF Gene

1) Based on E.coli ETCLOA, gene pykF is knocked out by adopting Red homologous recombination technology. Taking E.coli MG1655 genome as template, taking SEQ ID NO 17 and SEQ ID NO 18 as primer to carry out PCR amplification to obtain upstream homologous arm of pykF gene and purifying product. PCR amplification is carried out by taking SEQ ID NO 19 and SEQ ID NO 20 as primers to obtain the downstream homology arm of the pykF gene and purify the product.

2) PCR was performed using pKD3 as a template and SEQ ID NO 21 and SEQ ID NO 22 as primers to obtain a chloramphenicol resistance gene fragment.

3) And (3) obtaining a pykF gene knockout fragment by overlapping extension PCR amplification by using the PCR fragments obtained in the step 1) and the step 2) as templates and using SEQ ID NO 17 and SEQ ID NO 20 as primers. The gene knockout segment consists of an upstream homology arm gene segment, a chloramphenicol resistance gene segment and a downstream homology arm gene segment of a pykF gene.

4) And (3) transforming the gene knockout fragment into E.coli ETCLOA containing pKD46 plasmid by electric shock to obtain a positive transformant, and transforming the positive transformant into pCP20 plasmid to eliminate chloramphenicol resistance gene in the positive transformant to obtain lysA and pykF gene co-knockout bacteria E.coli ETCLOK.

Example 3 construction of genetically engineered Strain E.coli ETC15

(1) Taking the genome of E.coli MG1655 as a template, taking SEQ ID NO 23 and SEQ ID NO 24 as primers to carry out PCR amplification, obtaining a gene fragment fruK and carrying out PCR product purification, taking a plasmid pTrc99a as a template, taking SEQ ID NO 25 and SEQ ID NO 26 as primers to carry out PCR amplification, obtaining a promoter gene fragment Trc and carrying out PCR product purification. Carrying out overlap extension PCR amplification by using the purified Trc and fruK as templates and SEQ ID NO 25 and SEQ ID NO 24 as primers to obtain a gene fragment Trc-fruK and purifying a product. Plasmid pBAD33 was double digested with Sal I and Hind III and the product was purified. The obtained target gene fragment and the linearized vector are connected by a homologous recombination kit (Novozam, C112-1) to obtain a plasmid pBT-fruK.

(2) The gene fragments Trc-gapA, Trc-ppc, Trc-ask (M68V) were obtained in the same manner as in step 1). The fragments Trc-gapA-Trc-ppc-Trc-ask (M68V) were obtained by overlap PCR using purified Trc-gapA, Trc-ppc, and Trc-ask (M68V) as templates and purified. The plasmid pBT-fruK was digested simultaneously with Sam I and Xba I and the product was purified. The linearized pBT-fruK was ligated to the gene fragment Trc-gapA-Trc-ppc-Trc-ask (M68V) by means of a homologous recombination kit (Novozam, C112-1) to obtain the plasmid pBT-GPAF.

(3) Transforming the plasmid obtained in step 2) into e.coli ETC ok to obtain e.coli ETC 15.

Example 4 preparation of tetrahydropyrimidines by Shake flask fermentation

Carrying out shake flask fermentation by using genetically engineered bacterium E.coli ETC15, and specifically comprising the following steps:

(1) performing seed culture, namely inoculating the genetically engineered bacteria for producing tetrahydropyrimidine to a 500ml baffle shake flask with a seed culture medium after flat plate activation, wherein the inoculation amount is 1 percent, and performing shake culture at 37 ℃ and 200rpm for 10 hours;

(2) and (3) performing fermentation culture, namely inoculating the seed culture in the step (1) into a 500ml baffle shake flask filled with 50ml of fermentation culture in an inoculation amount of 10%, performing fermentation culture at 37 ℃ and 200rpm, and performing fermentation culture for 48 h.

The fermentation medium comprises the following components: 20g/L of yeast powder, 60g/L of jerusalem artichoke crude extract (inulin concentration), 10g/L of ammonium sulfate, 7g/L of glycerol, 5g/L of monopotassium phosphate, 1g/L of magnesium sulfate heptahydrate, 0.5g/L of sodium chloride, 0.1g/L of ampicillin sodium, 0.025g/L of chloramphenicol, 15g/L of basic magnesium carbonate and 0.2mM IPTG.

(3) Collecting fermentation liquor, centrifuging at 12000rpm, collecting supernatant, diluting by a certain multiple, filtering with 0.22 μm filter membrane, and performing liquid phase detection. Agilent 1260Infinity II liquid chromatography was used with a mobile phase of 2% acetonitrile, a flow rate of 0.5ml/min column TSKgel ODS-80TS (4.6X250mm,5 μm), a column temperature of 26 ℃, a dwell time of 20min, and a sample volume of 10 μ l.

(4) Three groups of parallel experiments can obtain that the yield of the tetrahydropyrimidine after 48 hours of shake flask fermentation is 19 +/-1.04 g/L.

EXAMPLE 5 preparation of tetrahydropyrimidines by fermentation in a fermenter

Taking genetically engineered escherichia coli E.coli ETC15 as a strain, inoculating the strain to an activation plate, culturing at 37 ℃ for 12-14h, scraping a single colony by using an inoculating loop, inoculating the single colony to a primary seed culture medium, culturing at 37 ℃ and 200rpm for 10-12h, inoculating to a secondary seed culture medium by using 1% of inoculation amount, and culturing at 37 ℃ and 200rpm for 8-10 h; transferring into a fermentation tank containing fermentation medium according to the inoculum size of 10% for fermentation culture, adding IPTG with final concentration of 0.2mM during fermentation culture for 4-6h, and supplementing Jerusalem artichoke crude extract with inulin concentration of 40% and ammonia water into the fermentation tank during fermentation.

Wherein, the growth is controlled by two different feeding modes in the fermentation culture process, in the growth stage, pH feedback feeding is adopted to reduce the generation of acetic acid, the feeding is started when the pH is more than 7.2, alkali liquor is not fed in the stage, the product synthesis stage is carried out after the target biomass is quickly reached, the fed-batch feeding is adopted in the stage, the concentration of residual sugar is controlled at 1g/L, the pH is adjusted to be 7.0-7.2 by 25 percent ammonia water, and the supply of sufficient carbon and nitrogen sources is ensured to prolong the product synthesis period. Wherein, the rotation speed, the ventilation capacity and the dissolved oxygen are linked in the fermentation culture process, the dissolved oxygen is controlled at 40 percent in the growth stage, and the dissolved oxygen is controlled at 20 percent in the product synthesis stage.

Fermentation medium: 15g/L of yeast powder, 15g/L of jerusalem artichoke crude extract (containing 15g/L of inulin), 15g/L of ammonium sulfate, 5g/L of glycerol, 4g/L of monopotassium phosphate, 1g/L of magnesium sulfate heptahydrate, 0.1g/L of trisodium citrate, 0.1g/L of manganese sulfate monohydrate, 0.1g/L of ferrous sulfate heptahydrate, 0.1g/L of ampicillin sodium and 0.025g/L of chloramphenicol.

Collecting fermentation liquor, centrifuging at 12000rpm, collecting supernatant, diluting by a certain multiple, filtering with 0.22 μm filter membrane, and performing liquid phase detection. Three groups of parallel experiments can obtain that the final yield of the tetrahydropyrimidine after fermentation for 48 hours in a fermentation tank is 58 +/-2.23 g/L.

The invention provides a thought and a method of a genetically engineered bacterium for high yield of tetrahydropyrimidine, and a plurality of methods and ways for implementing the technical scheme, and the above description is only a preferred embodiment of the invention, and it should be noted that, for those skilled in the art, a plurality of improvements and modifications can be made without departing from the principle of the invention, and these improvements and modifications should also be regarded as the protection scope of the invention. All the components not specified in the present embodiment can be realized by the prior art.

Sequence listing

<110> Nanjing university of industry

<120> genetic engineering bacterium for high yield of tetrahydropyrimidine and application thereof

<160> 26

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1263

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgccacatt cactgttcag caccgatacc gatctcaccg ccgaaaatct gctgcgtttg 60

cccgctgaat ttggctgccc ggtgtgggtc tacgatgcgc aaattattcg tcggcagatt 120

gcagcgctga aacagtttga tgtggtgcgc tttgcacaga aagcctgttc caatattcat 180

attttgcgct taatgcgtga gcagggcgtg aaagtggatt ccgtctcgtt aggcgaaata 240

gagcgtgcgt tggcggcggg ttacaatccg caaacgcacc ccgatgatat tgtttttacg 300

gcagatgtta tcgatcaggc gacgcttgaa cgcgtcagtg aattgcaaat tccggtgaat 360

gcgggttctg ttgatatgct cgaccaactg ggccaggttt cgccagggca tcgggtatgg 420

ctgcgcgtta atccggggtt tggtcacgga catagccaaa aaaccaatac cggtggcgaa 480

aacagcaagc acggtatctg gtacaccgat ctgcccgccg cactggacgt gatacaacgt 540

catcatctgc agctggtcgg cattcacatg cacattggtt ctggcgttga ttatgcccat 600

ctggaacagg tgtgtggtgc tatggtgcgt caggtcatcg aattcggtca ggatttacag 660

gctatttctg cgggcggtgg gctttctgtt ccttatcaac agggtgaaga ggcggttgat 720

accgaacatt attatggtct gtggaatgcc gcgcgtgagc aaatcgcccg ccatttgggc 780

caccctgtga aactggaaat tgaaccgggt cgcttcctgg tagcgcagtc tggcgtatta 840

attactcagg tgcggagcgt caaacaaatg gggagccgcc actttgtgct ggttgatgcc 900

gggttcaacg atctgatgcg cccggcaatg tacggtagtt accaccatat cagtgccctg 960

gcagctgatg gtcgttctct ggaacacgcg ccaacggtgg aaaccgtcgt cgccggaccg 1020

ttatgtgaat cgggcgatgt ctttacccag caggaagggg gaaatgttga aacccgcgcc 1080

ttgccggaag tgaaggcagg tgattatctg gtactgcatg atacaggggc atatggcgca 1140

tcaatgtcat ccaactacaa tagccgtccg ctgttaccag aagttctgtt tgataatggt 1200

caggcgcggt tgattcgccg tcgccagacc atcgaagaat tactggcgct ggaattgctt 1260

taa 1263

<210> 2

<211> 1413

<212> DNA

<213> nucleotide Sequence encoding gene pykF (Artificial Sequence)

<400> 2

atgaaaaaga ccaaaattgt ttgcaccatc ggaccgaaaa ccgaatctga agagatgtta 60

gctaaaatgc tggacgctgg catgaacgtt atgcgtctga acttctctca tggtgactat 120

gcagaacacg gtcagcgcat tcagaatctg cgcaacgtga tgagcaaaac tggtaaaacc 180

gccgctatcc tgcttgatac caaaggtccg gaaatccgca ccatgaaact ggaaggcggt 240

aacgacgttt ctctgaaagc tggtcagacc tttactttca ccactgataa atctgttatc 300

ggcaacagcg aaatggttgc ggtaacgtat gaaggtttca ctactgacct gtctgttggc 360

aacaccgtac tggttgacga tggtctgatc ggtatggaag ttaccgccat tgaaggtaac 420

aaagttatct gtaaagtgct gaacaacggt gacctgggcg aaaacaaagg tgtgaacctg 480

cctggcgttt ccattgctct gccagcactg gctgaaaaag acaaacagga cctgatcttt 540

ggttgcgaac aaggcgtaga ctttgttgct gcttccttta ttcgtaagcg ttctgacgtt 600

atcgaaatcc gtgagcacct gaaagcgcac ggcggcgaaa acatccacat catctccaaa 660

atcgaaaacc aggaaggcct caacaacttc gacgaaatcc tcgaagcctc tgacggcatc 720

atggttgcgc gtggcgacct gggtgtagaa atcccggtag aagaagttat cttcgcccag 780

aagatgatga tcgaaaaatg tatccgtgca cgtaaagtcg ttatcactgc gacccagatg 840

ctggattcca tgatcaaaaa cccacgcccg actcgcgcag aagccggtga cgttgcaaac 900

gccatcctcg acggtactga cgcagtgatg ctgtctggtg aatccgcaaa aggtaaatac 960

ccgctggaag cggtttctat catggcgacc atctgcgaac gtaccgaccg cgtgatgaac 1020

agccgtctcg agttcaacaa tgacaaccgt aaactgcgca ttaccgaagc ggtatgccgt 1080

ggtgccgttg aaactgctga aaaactggat gctccgctga tcgtggttgc tactcagggc 1140

ggtaaatctg ctcgcgcagt acgtaaatac ttcccggatg ccaccatcct ggcactgacc 1200

accaacgaaa aaacggctca tcagttggta ctgagcaaag gcgttgtgcc gcagcttgtt 1260

aaagagatca cttctactga tgatttctac cgtctgggta aagaactggc tctgcagagc 1320

ggtctggcac acaaaggtga cgttgtagtt atggtttctg gtgcactggt accgagcggc 1380

actactaaca ccgcatctgt tcacgtcctg taa 1413

<210> 3

<211> 2451

<212> DNA

<213> nucleotide Sequence of the coding Gene ectABC Gene (Artificial Sequence)

<400> 3

atgagcacgc caataatacc ttttacccct tctgcagacc ttgctcgccc aacagtcgct 60

gatgctgtgg ttgggcatgc gtctactcca ttattcattc gcaagccaaa tgcagatgat 120

ggttggggca tctacgagtt gataaaagct tgcccgccgc ttgatgttaa ttctgcttat 180

gcttatctac tgctggcaac ccagtttcgt gacacctgcg cggtggcgac aaatgaagag 240

ggtgagatag ttgggtttgt atcgggctat gtgaaagaca atgcgccgga cacgtacttt 300

ctatggcagg ttgccgttgg tgaaaaagca cgtggtactg gcttggcacg ccgtttagtc 360

gaagccatta tgtcgcgccc agaacttgat aatgtgcatc acctggaaac gacgatcacg 420

cccgacaatc aggcctcttg gggtctattt cgccgcctcg ctgcccgttg gcaagccccg 480

ttaaacagcc gtgaatattt ctctaccgaa caactcggtg gagagcatga tcctgaaaac 540

ctagttcgta taggtccatt tcaaacagac agcatgtaac cgaacgaacc aactcttgcc 600

caatgcgcgg ctcttgctag ctggttacgt tttaaattgg ttgccgctgt ttccgccagt 660

tcgtcccacg cttgactcaa tgatttaaaa aggaggtcgt taatgcagac ccaaacgctt 720

gaacgcatgg aatctaacgt acgtacttac tcgcgctcat tccccgttgt atttaccaaa 780

gcacagaatg ctcgtttaac ggatgagaat ggtcgtgagt atattgattt cctagccggt 840

gccggcacgc tcaattacgg tcacaataac ccacatctca aacaggcaat gatcgactat 900

ctgtcaacag atggcattgt tcatggcctg gatatgtgga ccgcggcaaa gcgcgattac 960

ctagaaaccc ttgaagaagt tatcttcaaa ccgcgtggct tagactacaa ggtacacctg 1020

ccgggcccga ccggaactaa cgcagtggaa gcggccatcc gtttggcccg tgttgctaaa 1080

ggtcgccata acattgtgac ctttaccaac ggcttccacg gcgtcaccat gggagcactg 1140

gcaaccacag gtaaccgcaa attccgcgaa gcgaccggcg gcattcccac tcagggtgca 1200

agctttttac cctttgatgg ttatatggga gagcatgcag atacgctgga ttacttcgaa 1260

aaattgctta acgataaatc tggcggcctc gatatacccg cgggtgttat tgtcgaaacc 1320

gtgcaaggcg agggcggtat taacgtagca ggcctagagt ggctcaagcg tctagaaagc 1380

atctgtcgcg cccatgatat tctgttgatc atcgacgaca tccaggcagg ctgcggccgc 1440

acgggcaagt tctttagctt tgaacacgcg ggcatcacgc ccgatatcgt gacgaattca 1500

aaatcacttt ctggattcgg actgccattt gcccatgtat tgatgcgtcc agaacttgat 1560

aaatggaagc ctggtcagta caacggcacg ttccgtggct tcaacttggc gatggtgacc 1620

gccacggccg cacttaaaaa atattggtcc aacgatgttt ttgagcgtga tgttcagcgc 1680

aaagcgcgta ttgttgaaga gcgtttccaa aagctagcgg ccttactcag cgagaacggc 1740

atgcctgcca ctgagcgtgg acgtggcctc atgcgcggaa ttgatgttgt ctcaggcgat 1800

attgctgaca agatcaccag caaagcattt gagcacggcc ttattattga gaccagcggc 1860

caagacggcg aagtcgtcaa atgcctatgc ccgctgacca tcagtgatga ggacttgtta 1920

gaagcgctgg atattctaga agcctctgtc aatgctgtta tccaagcgtg atttagcgtt 1980

aggctaattc agtagcatca attcagcact gggccgcgtt gattaatact aagtgcctca 2040

taacggagca attctatgat cgttcgtaac cttgaagaag cacgtaaaac agatcgtctt 2100

gtcaccgccg agaatggcaa ctgggacagc acgcgtcttg ttctagccaa tgataatgcg 2160

ggtttttcat ttcatatcac ccgtattttc ccaggcactg agacgcatat tcattacaaa 2220

aatcactatg aagctgtgtt ttgctatgaa ggagaaggcg aggtagaaac cttggccgat 2280

ggcaaaatct ggccgatcaa agcaggcgat atttatctgc tggatcagca cgacgaacac 2340

ctgttacgcg gtaaagaaaa aggcatgacc gttgcgtgtg tatttactcc cgctatcact 2400

ggcaacgaag tacaccagga agatggctca tacgccgcac ctactgggtg a 2451

<210> 4

<211> 1470

<212> DNA

<213> nucleotide Sequence (Artificial Sequence) encoding gene cscA

<400> 4

atggatagaa ttcagcaggc ggaagaagcc ttgaagaaag ccgagggtaa agtgaaacaa 60

agatatcggc tggggtacca tattatgccc cgggcgaatt ggatcaatga tccgaacgga 120

cttattcagt ttaagggaga ataccacgtt ttttttcagc atcatccgtt tgatgagaac 180

tgggggccga tgcattgggg gcatgtaaag agtaaagacc tcattcattg ggagcacttg 240

ccgattgcct tagcgccggg cgacgcattt gatcaaagcg gctgtttttc gggaagcgcg 300

gtcgatgatc gtggaagact tgtcttaatc tataccgggc ataacatgat tgaccccgag 360

aaagaccttt tctatcaaac tcaaaatatc gctgtcagcc aagatggtgc agtgtttgaa 420

aagcttcaag ataaccctgt tattgcggaa ccgccggaag acagctcccg ccattttcgc 480

gacccgaaag tgtggaagca tcgcggagac tggtatatgg tagtcggcaa ttccacaaaa 540

gaaaacgtcg ggcgggtcat tttataccgt tcatctgatt tgcgtaattg ggaatacgca 600

ggtgttctcg cccaaagtga cggtcatctc ggctatatgt gggaatgtcc tgatttcttt 660

gaattaggcg gcaaacatgt cctgctaatt tcgccacagg gtattgaagc cgacggtgat 720

tcctataaaa atttacatca aaccggttat ttaattggtg actataatga tgaaacaaat 780

aaatttacac acggcgcttt taaagaactg gatcacggcc atgactttta cgccgttcaa 840

acattactgg atgataaagg acgcagaatt gccgtcggct ggatggatat gtgggaatcc 900

gagatgccga caaaagcgga cggatggtgc ggtgctttga cactgccgcg ggaactgaca 960

ttgcgtgatg atcataaact tttgatgaat cccgtggaag aaaccaagca gctgcgaaaa 1020

atgaaatatc gggaatgtgc cggccggtcc gtttcaggca gttacttggc aaagacatcc 1080

gaagagctgc tggaagtcca agtcgtgtat gatgtaaacg attgcgatgc cgaaacggca 1140

ggtattaaaa ttcgcggcct tgatgaagag gaacttgtgc ttaagtacaa tctaacggat 1200

aaaaaattga cacttgattg caccaagatg gggaaagcga aagacggtgt gagaagagtg 1260

cggatggatg caagcggcaa gctggcgctg cgaatattta ttgacaggtc ttcgattgaa 1320

gtattcgcca atcatggaga agcaacgatg acaagccgta tctatccgaa agagggcaga 1380

ttggggcttg agctgttttc tgagaaaggc gctgtaaagg ttgaggaatt cacctattgg 1440

acgttaaaag acatttggaa aaaaagctaa 1470

<210> 5

<211> 1272

<212> DNA

<213> encoding gene ask (nucleotide Sequence of M68V Artificial Sequence)

<400> 5

gtgggccttg tcgtgcagaa gtacggcggc tcatccgttg cggatgccga gggcatcaag 60

cgcgttgcca agcgaatcgt cgaggccaag aagaacggca accaggttgt cgtcgtggtc 120

tcggcgatgg gcgacacgac ggacgagctg atcgatctcg cgcaggaagt gtccccgatc 180

ccgtcgggac gcgagttcga cgtgctgctg accgccggag agcggatctc catggccctg 240

ctggcgatgg cgatcaaaaa cctcggccat gaggcgcagt ccttcaccgg tagccaggcc 300

ggtgtgatca ctgactccgt gcacaacaag gcacggatca tcgatgtcac gccgggccgc 360

atcaaggcgt ccctcgacga gggcaacatc gccatcgtcg ccggattcca gggcgtgtcc 420

caggacaaga aggacatcac gacgctcggg cgcggtggtt cggacaccac cgcggtggcg 480

ctcgccgccg ccctgaacgc cgatgtctgc gagatctaca ccgacgtcga cggcgtcttc 540

accgccgacc cgcgggtcgt gaagaaggcc cggaagatcg agtggatctc cttcgaggac 600

atgctggagc tggccagctc cggctccaag gtgctgctgc accgctgcgt cgagtacgca 660

cgccgttaca acatcccgat ccacgtccgc tcctccttct cggggctgca gggcacatgg 720

gtcagcaacg aaccgcaagg ggacaggccg atggaacagg cgatcatctc gggcgtcgca 780

cacgacacct ccgaggcgaa ggtcacggtc gtcggggtcc cggacaagcc gggcgaggcc 840

gcgcggatct tccgtgccat cgccgactcc gaggtcaaca tcgacatggt cgtccagaac 900

gtgtcggcgg cctccaccgg tctgacggac atctccttca cgctgccgaa ggccgagggc 960

cgcaaggccg tcgccgcgct ggagaagacc cgggccgcgg tcggcttcga ctcgctccgc 1020

tacgacgacc agatcgccaa gatctcgctg gtcggcgcgg gcatgaagac caaccccggc 1080

gtcaccgcga cgttcttcga ggcgctgtcg aacgcgggcg tgaacatcga gctcatctcg 1140

acctccgaga tccgcatctc ggtcgtcacc cgtgccgatg acgtcaacga ggccgtccag 1200

gcggtgcaca gcgccttcgg cctcgacagt gagaccgacg aagcagtcgt ctacggcggc 1260

accgggcgat ga 1272

<210> 6

<211> 939

<212> DNA

<213> nucleotide Sequence encoding Gene fruK (Artificial Sequence)

<400> 6

atgagcagac gtgttgctac tatcaccctt aatccggctt atgaccttgt tggtttctgc 60

ccggaaattg aacgcggcga agtgaacctg gtgaaaacca ccggtctgca tgcggcgggt 120

aaaggcatca acgtggccaa agtattaaaa gacctgggaa ttgatgtcac cgttggcggc 180

ttcctgggta aagacaatca ggatggtttt cagcaactgt tcagcgagct gggcattgcc 240

aaccgtttcc aggttgtaca ggggcgcacc cgaattaacg ttaagctgac ggaaaaagac 300

ggcgaagtga ccgacttcaa cttctcgggt tttgaagtca cccccgccga ctgggaacgc 360

tttgtgactg attctctgag ctggctcggt cagttcgata tggtctgtgt cagcggaagc 420

ttaccgtcag gcgtcagccc ggaagcgttc accgactgga tgactcgcct gcgtagtcag 480

tgtccttgca ttatctttga tagtagccgt gaagcgttag tagcaggttt gaaagcggca 540

ccgtggctgg tgaaacctaa ccgccgcgag ctggaaatct gggcaggccg taaactgcct 600

gaaatgaaag atgtgattga agctgcacat gcgctacgtg aacaaggcat cgcgcatgtt 660

gttatttcac tgggtgccga aggcgcgctt tgggttaatg cctccggcga atggatcgcc 720

aaaccaccgt cagtcgatgt cgtaagcacc gttggcgcag gggattctat ggttggtggc 780

ctgatttatg gcttgctgat gcgtgaatcc agtgaacaca cactgcgtct ggcgacagct 840

gttgcagccc tggcggtaag tcaaagcaat gtgggtatta ccgatcgtcc gcagttggcc 900

gcaatgatgg cgcgcgtcga cttacaacct tttaactga 939

<210> 7

<211> 996

<212> DNA

<213> nucleotide Sequence encoding gapA Gene (Artificial Sequence)

<400> 7

atgactatca aagtaggtat caacggtttt ggccgtatcg gtcgcattgt tttccgtgct 60

gctcagaaac gttctgacat cgagatcgtt gcaatcaacg acctgttaga cgctgattac 120

atggcataca tgctgaaata tgactccact cacggccgtt tcgacggtac cgttgaagtg 180

aaagacggtc atctgatcgt taacggtaaa aaaatccgtg ttaccgctga acgtgatccg 240

gctaacctga aatgggacga agttggtgtt gacgttgtcg ctgaagcaac tggtctgttc 300

ctgactgacg aaactgctcg taaacacatc accgctggtg cgaagaaagt ggttatgact 360

ggtccgtcta aagacaacac tccgatgttc gttaaaggcg ctaacttcga caaatatgct 420

ggccaggaca tcgtttccaa cgcttcctgc accaccaact gcctggctcc gctggctaaa 480

gttatcaacg ataacttcgg catcatcgaa ggtctgatga ccaccgttca cgctactacc 540

gctactcaga aaaccgttga tggcccgtct cacaaagact ggcgcggcgg ccgcggcgct 600

tcccagaaca tcatcccgtc ctctaccggt gctgctaaag ctgtaggtaa agtactgcca 660

gaactgaatg gcaaactgac tggtatggcg ttccgcgttc cgaccccgaa cgtatctgta 720

gttgacctga ccgttcgtct ggaaaaagct gcaacttacg agcagatcaa agctgccgtt 780

aaagctgctg ctgaaggcga aatgaaaggc gttctgggct acaccgaaga tgacgtagta 840

tctaccgatt tcaacggcga agtttgcact tccgtgttcg atgctaaagc tggtatcgct 900

ctgaacgaca acttcgtgaa actggtatcc tggtacgaca acgaaaccgg ttactccaac 960

aaagttctgg acctgatcgc tcacatctcc aaataa 996

<210> 8

<211> 2652

<212> DNA

<213> nucleotide Sequence encoding Gene ppc (Artificial Sequence)

<400> 8

atgaacgaac aatattccgc attgcgtagt aatgtcagta tgctcggcaa agtgctggga 60

gaaaccatca aggatgcgtt gggagaacac attcttgaac gcgtagaaac tatccgtaag 120

ttgtcgaaat cttcacgcgc tggcaatgat gctaaccgcc aggagttgct caccacctta 180

caaaatttgt cgaacgacga gctgctgccc gttgcgcgtg cgtttagtca gttcctgaac 240

ctggccaaca ccgccgagca ataccacagc atttcgccga aaggcgaagc tgccagcaac 300

ccggaagtga tcgcccgcac cctgcgtaaa ctgaaaaacc agccggaact gagcgaagac 360

accatcaaaa aagcagtgga atcgctgtcg ctggaactgg tcctcacggc tcacccaacc 420

gaaattaccc gtcgtacact gatccacaaa atggtggaag tgaacgcctg tttaaaacag 480

ctcgataaca aagatatcgc tgactacgaa cacaaccagc tgatgcgtcg cctgcgccag 540

ttgatcgccc agtcatggca taccgatgaa atccgtaagc tgcgtccaag cccggtagat 600

gaagccaaat ggggctttgc cgtagtggaa aacagcctgt ggcaaggcgt accaaattac 660

ctgcgcgaac tgaacgaaca actggaagag aacctcggct acaaactgcc cgtcgaattt 720

gttccggtcc gttttacttc gtggatgggc ggcgaccgcg acggcaaccc gaacgtcact 780

gccgatatca cccgccacgt cctgctactc agccgctgga aagccaccga tttgttcctg 840

aaagatattc aggtgctggt ttctgaactg tcgatggttg aagcgacccc tgaactgctg 900

gcgctggttg gcgaagaagg tgccgcagaa ccgtatcgct atctgatgaa aaacctgcgt 960

tctcgcctga tggcgacaca ggcatggctg gaagcgcgcc tgaaaggcga agaactgcca 1020

aaaccagaag gcctgctgac acaaaacgaa gaactgtggg aaccgctcta cgcttgctac 1080

cagtcacttc aggcgtgtgg catgggtatt atcgccaacg gcgatctgct cgacaccctg 1140

cgccgcgtga aatgtttcgg cgtaccgctg gtccgtattg atatccgtca ggagagcacg 1200

cgtcataccg aagcgctggg cgagctgacc cgctacctcg gtatcggcga ctacgaaagc 1260

tggtcagagg ccgacaaaca ggcgttcctg atccgcgaac tgaactccaa acgtccgctt 1320

ctgccgcgca actggcaacc aagcgccgaa acgcgcgaag tgctcgatac ctgccaggtg 1380

attgccgaag caccgcaagg ctccattgcc gcctacgtga tctcgatggc gaaaacgccg 1440

tccgacgtac tggctgtcca cctgctgctg aaagaagcgg gtatcgggtt tgcgatgccg 1500

gttgctccgc tgtttgaaac cctcgatgat ctgaacaacg ccaacgatgt catgacccag 1560

ctgctcaata ttgactggta tcgtggcctg attcagggca aacagatggt gatgattggc 1620

tattccgact cagcaaaaga tgcgggagtg atggcagctt cctgggcgca atatcaggca 1680

caggatgcat taatcaaaac ctgcgaaaaa gcgggtattg agctgacgtt gttccacggt 1740

cgcggcggtt ccattggtcg cggcggcgca cctgctcatg cggcgctgct gtcacaaccg 1800

ccaggaagcc tgaaaggcgg cctgcgcgta accgaacagg gcgagatgat ccgctttaaa 1860

tatggtctgc cagaaatcac cgtcagcagc ctgtcgcttt ataccggggc gattctggaa 1920

gccaacctgc tgccaccgcc ggagccgaaa gagagctggc gtcgcattat ggatgaactg 1980

tcagtcatct cctgcgatgt ctaccgcggc tacgtacgtg aaaacaaaga ttttgtgcct 2040

tacttccgct ccgctacgcc ggaacaagaa ctgggcaaac tgccgttggg ttcacgtccg 2100

gcgaaacgtc gcccaaccgg cggcgtcgag tcactacgcg ccattccgtg gatcttcgcc 2160

tggacgcaaa accgtctgat gctccccgcc tggctgggtg caggtacggc gctgcaaaaa 2220

gtggtcgaag acggcaaaca gagcgagctg gaggctatgt gccgcgattg gccattcttc 2280

tcgacgcgtc tcggcatgct ggagatggtc ttcgccaaag cagacctgtg gctggcggaa 2340

tactatgacc aacgcctggt agacaaagca ctgtggccgt taggtaaaga gttacgcaac 2400

ctgcaagaag aagacatcaa agtggtgctg gcgattgcca acgattccca tctgatggcc 2460

gatctgccgt ggattgcaga gtctattcag ctacggaata tttacaccga cccgctgaac 2520

gtattgcagg ccgagttgct gcaccgctcc cgccaggcag aaaaagaagg ccaggaaccg 2580

gatcctcgcg tcgaacaagc gttaatggtc actattgccg ggattgcggc aggtatgcgt 2640

aataccggct aa 2652

<210> 9

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atggaattcg agctcggtac catgagcacg ccaataatac cttt 44

<210> 10

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

caggtcgact ctagaggatc ctcacccagt aggtgcggcg 40

<210> 11

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gaccagatct atggatagaa ttcagcaggc gg 32

<210> 12

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gcttgcatgc ctgcaggtcg acttagcttt ttttccaaat gtcttttaac gtcca 55

<210> 13

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cgcacctact gggtgaggat ccttgacaat taatcatccg gctcgtataa tgt 53

<210> 14

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

attctatcca tagatctggt ctgtttcctg tgtgaaa 37

<210> 15

<211> 71

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

caaacgcacc ccgatgatat tgtttttacg gcagatgtta tcgatcaggc ggtgtaggct 60

ggagctgctt c 71

<210> 16

<211> 71

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cggttcaatt tccagtttca cagggtggcc caaatggcgg gcgatttgct cacatgggaa 60

ttagccatgg t 71

<210> 17

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ttgcgtaacc ttttccctgg aacgt 25

<210> 18

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gaagcagctc cagcctacac ttcagattcg gttttcggtc cgatg 45

<210> 19

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

accatggcta attcccatgt tgcactggta ccgagcggca ctact 45

<210> 20

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

acccaggatt accgcgccca gtacc 25

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gtgtaggctg gagctgcttc 20

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

acatgggaat tagccatggt 20

<210> 23

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gaccagatct atgagcagac gtgttgctac tatcac 36

<210> 24

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

atccgccaaa acagccaagc tttcagttaa aaggttgtaa gtcgacgc 48

<210> 25

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ccggggatcc tctagagtcg acttgacaat taatcatccg gctcgtataa tgt 53

<210> 26

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cgtctgctca tagatctggt ctgtttcctg tgtgaaa 37

Claims

1. The genetically engineered bacterium for high yield of tetrahydropyrimidine is E.coli ETC15, and the host cell of the genetically engineered bacterium is E.coli MG1655, which comprises: two genes, lysA and pykF, are defective; ectoine synthesis gene cluster ectABC gene controlled by trc promoter; an inulin exonuclease cscA gene controlled by the trc promoter; ask from streptomyces albus under the control of trc promoter and fruK, gapA and ppc from escherichia coli under the control of trc promoter; wherein the nucleotide sequence of the encoding gene lysA is shown as a sequence table SEQ ID NO. 1;

the nucleotide sequence of the coding gene pykF is shown as a sequence table SEQ ID NO. 2;

the nucleotide sequence of the coding gene ectABC gene is shown as a sequence table SEQ ID NO. 3;

the nucleotide sequence of the coding gene ask is shown as a sequence table SEQ ID NO. 5;

2. The genetically engineered bacterium of claim 1, wherein the ecto-pyrimidine synthesis gene cluster ectABC gene is derived from any one of Halomonas meibomiae, Halobacterium halodurans, Halomonas elongata or Cladosporium sp; the inulase cscA gene is derived from bacillus amyloliquefaciens.

3. The method for constructing a genetically engineered bacterium according to any one of claims 1 to 2, comprising the steps of:

(1) cloning a tetrahydropyrimidine synthesis gene cluster ectABC and an inulin exonuclease gene cscA by taking a pTrc99a plasmid as a vector to obtain a recombinant plasmid named as pTETCsA, wherein the two genes are controlled by independent trc promoters, and the recombinant plasmid is transformed into Escherichia coli E.coli MG1655 to construct an initial strain capable of producing tetrahydropyrimidine by using the jerusalem artichoke crude extract, and named as E.coli ETCO;

(2) knocking out lysA and pykF which encode diaminopimelate decarboxylase and pyruvate kinase in E.coli ETCO, and naming E.coli ETCK;

(3) the method comprises the steps of taking pBAD33 plasmid as a carrier to serially express fruK, gapA and ppc from escherichia coli and ask from streptomyces albus to obtain a recombinant plasmid pBT-GPAF, controlling the four genes by independent trc promoters, converting the recombinant plasmid into escherichia coli E.coli ETOK, and constructing a strain which finally utilizes the jerusalem artichoke crude extract to produce tetrahydropyrimidine at high yield and is named as E.coli ETC 15.

4. The use of the genetically engineered bacterium of claim 1 for the preparation of tetrahydropyrimidine.

5. The use according to claim 4, wherein the step of preparing tetrahydropyrimidine by shake flask fermentation is as follows:

6. Use according to claim 5, characterized in that: the fermentation medium in the step (2) comprises the following components: 5-20g/L of organic nitrogen source, 50-150g/L of carbon source, 5-20g/L of inorganic nitrogen source, 5-10g/L of glycerol, 2-8g/L of monopotassium phosphate, 0.5-1g/L of magnesium sulfate heptahydrate, 0.5-1g/L of sodium chloride, 0.1g/L of ampicillin sodium, 0.025g/L of chloramphenicol, and 5-20g/L, IPTG 0.1.1-1 mM of basic magnesium carbonate.

7. The use according to claim 4, wherein the process for the fermentative preparation of tetrahydropyrimidine in fermenters comprises the following steps:

8. The use of claim 7, wherein the growth is controlled by two different feeding modes in the fermentation culture process, in the growth stage, pH feedback feeding is adopted to reduce the generation of acetic acid, feeding is started at pH6.5-7.5, alkali liquor is not fed in the stage, and dissolved oxygen is controlled to be 40% -50%; and (3) entering a product synthesis stage after the target biomass is quickly reached, wherein fed-batch materials are adopted in the product synthesis stage, the concentration of residual sugar is controlled to be 1-5g/L, the pH value is adjusted to be 7.0-8.0 by 20-30% ammonia water, and the dissolved oxygen is controlled to be 10% -20%.

9. The use of claim 7, wherein the fermentation medium components comprise: 5-20g/L of organic nitrogen source, 1-20g/L of carbon source, 5-20g/L of inorganic nitrogen source, 5-10g/L of glycerol, 2-4g/L of monopotassium phosphate, 0.5-1g/L of magnesium sulfate heptahydrate, 0.05-0.1g/L of trisodium citrate, 0.05-0.1g/L of manganese sulfate monohydrate, 0.05-0.1g/L of ferrous sulfate heptahydrate, 0.1g/L of ampicillin sodium, and 0.025g/L, IPTG 0.1.1-1 mM of chloramphenicol.