CN112011583A

CN112011583A - Method for synthesizing 2' -FL in escherichia coli by utilizing microbial quorum sensing and application

Info

Publication number: CN112011583A
Application number: CN202010946953.2A
Authority: CN
Inventors: 刘振云; 程丰伟; 郭涛; 王旭; 倪磊
Original assignee: Suzhou Yixi Biotechnology Co ltd
Current assignee: Suzhou Yixi Biotechnology Co ltd
Priority date: 2020-09-10
Filing date: 2020-09-10
Publication date: 2020-12-01

Abstract

The invention provides an application of microbial quorum sensing, which applies the microbial quorum sensing to the synthesis of 2 '-fucosyllactose (2' -FL) through self-induction in a genome integration mode. The invention also provides a method for inducing expression of related protein in a 2 '-FL synthesis pathway in microbial quorum sensing escherichia coli and synthesizing 2' -FL. In the method for synthesizing the 2' -FL, a self-induced gene path is constructed by using a promoter and a structural gene corresponding to Vibrio fischeri quorum sensing, wherein the self-induced gene path is luxpL-RBS-luxI-TT-luxpL-RBS-luxR-TT-luxpR-RBS-X-TT, and X is a CDS sequence of a gene to be expressed. The method integrates the quorum sensing mode of the microorganisms into the genome of the escherichia coli, expresses related genes in the 2 '-FL synthesis path, enables the escherichia coli to obtain a positive feedback mechanism, and can automatically start in-vivo synthesis of the 2' -FL when the density reaches a certain threshold value.

Description

Method for synthesizing 2' -FL in escherichia coli by utilizing microbial quorum sensing and application

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to synthesis of 2' -FL.

Background

1. Quorum sensing

Quorum sensing (Quorum sensing) is a system of stimuli and responses that are correlated with population density. Many bacteria regulate gene expression according to the size of their population by quorum sensing.

The signalling molecules produced and secreted by bacteria by quorum sensing are commonly referred to as autoinducers. These bacteria also have a receptor that can detect specific signal molecules (inducers). When the inducer is attached to the receptor, it allows the transcription mechanism of certain genes to be activated, including those genes that are used to synthesize the inducer. There are few bacteria that can detect the self-secreted inducing substances. Therefore, in order to activate the transcription mechanism of a specific gene, the cell must be bound to other signal substances secreted from other cells.

Introduction to HMO

2.1HMOs biological function

(1) Maintaining microecological balance of the intestinal tract

(2) Protection against pathogenic infection

(3) Modulating immunity

(4) Inhibiting inflammatory response

(5) Promoting brain development

2.2 regulatory policy background

Currently, there are two types of human milk oligosaccharides approved for use in infant formulas: 2-fucosyllactose (2' -FL) and Lacto-N-neotetraose (Lacto-N-neotame).

One of the reasons why domestic HMOs products are not marketed is that HMOs have not been incorporated into new food additive varieties. However, the national food safety risk assessment Center (CFSA) has already publicly requested opinions to society in 2016 (8 months), and the regulations on the use range, dosage, quality specification requirements, production process and technical requirements and the like of HMOs as a new food additive variety are included.

Whereas in europe, 11 months 2015, the european union food safety agency (EFSA) issued opinions on the safety of LNnt and 2' -FL as novel food ingredients for use as a food supplement for children; the european union, 3 months 2016, approved LNnt and 2' -FL for use as a new food ingredient; in the same year, the FDA in the united states approved 2' -FL and LNnt as food additives to be added to foods in 5 months and 8 months, respectively, and specified the maximum addition amount and the range of use and the like of these two additives.

2017/2470 approved by eu regulation-the unified listing of new eu food ingredients lists six items for 2' -FL and LNnT, including: the range of food products includes infant formulas, follow-up formulas, infant-directed milk-based drinks and similar products, non-flavored pasteurized or sterilized (including UHT high temperature flash sterilization) milk-based products, cereal-based processed foods, infant-directed foods for infants and foods for special medical uses.

22/7/2019, published by the australia new food standards institute (FSANZ) No. 85-19, of which application No. 1155 is intended to approve the use of 2' -FL and LNnT in infant formulas and other products and specifies: if only 2 '-FL, 2' -FL is added, the concentration is not more than 96mg/100 kJ; ② if 2 '-FL and LNnT are added, LNnT does not exceed 24mg/100kJ, and the sum of 2' -FL and LNnT does not exceed 96mg/100 kJ.

At present, countries and regions such as hong Kong special administrative districts, Pakistan, Singapore, Malaysia, Vietnam and New Zealand in Asia-Tai region successively pass regulatory approval.

Due to the above problems, in contrast, there are few domestic studies on HMOs and many foreign related research papers. At present, there are many synthetic methods for human milk oligosaccharides, which are mainly classified into chemical synthesis methods and biological synthesis methods. The biosynthesis methods are divided into enzymatic synthesis methods and cell fermentation synthesis methods. Among them, the enzymatic synthesis method is expensive, requires purification of various enzymes, and is not favorable for industrial mass production of HMOs. Currently, the biosynthesis method becomes the method for synthesizing HMO which is most likely to realize industrial production.

Compared with the method of extracting enzyme participating in oligosaccharide synthesis in microbial cells, adding the extracted enzyme into a reaction system and directly carrying out oligosaccharide synthesis in the microbial cells by utilizing the enzyme itself or transferred enzyme by genetic engineering, the method is a cell fermentation synthesis method. The method utilizes glycosyltransferase for in vivo synthesis without enzyme purification, and sugar nucleotide donor can be generated by cell metabolism, thereby greatly reducing cost.

Among them, 2-fucosyllactose (2' -FL) is one of the main components of HMO and is also one of the components currently approved for production in foreign countries for use in food additives.

For the intracellular mass synthesis of 2' -FL, the activity of fucosyltransferase and the amount of its substrate GDP-L-fucose are bottlenecks in the product synthesis. Escherichia coli is capable of endogenously synthesizing GDP-L-fucose. GDP-L-fucose is an intermediate in the biosynthesis of kojic acid, a cell wall component. Two pathways for GDP-L-fucose production: a de novo synthetic pathway branching from the glycolytic intermediate fructose-6-phosphate, and a salvage synthetic pathway requiring L-fucose.

Along the de novo synthesis pathway of 2' -FL, β -D-mannose-1-phosphate is formed from fructose-6-phosphate by mannose-6-phosphate isomerase (Man A) and phosphomannose mutase (Man B). Mannose-1-phosphate guanylyltransferase (Man C) catalyzes the nucleotide transfer from GTP to alpha-D-mannose-1-phosphate to produce GDP-mannose, which is then converted into GDP-L-fucose in two parts catalyzed by GDP-mannose 6-dehydrogenase (Gmd) and NADPH-dependent GDP-L-fucose synthase (Wcag), respectively. Under non-engineered expression, the biosynthetic amount of GDP-L-fucose production may be too low to allow fucosylation with co-expressed fucosyltransferases. In addition, on this basis, there is still a need to express Fucosyltransferase (FUT) for the final synthesis of 2' -FL.

In another aspect, cytoplasmic L-fucose is converted to GDP-L-fucose by the sequential action of fucose 1-kinase and fucose-1-P guanylyltransferase in a GDP-L-fucose salvage synthesis pathway. The flux through the salvage synthesis pathway can be increased by additionally expressing the fkp gene and knocking out the fucose isomerase (FucI) and fucose kinase (FucK) genes.

The current method is to use plasmid vector to make complementary expression and over-expression of the above-mentioned related genes, the commonly used promoters with strong prokaryotic expression are T7lac, Ptac, etc. which can be induced by isopropyl-beta-D-thiogalactoside (IPTG), which is a laboratory toxic agent and can cause damage when inhaled, ingested or absorbed by skin.

The drawbacks of the current methods are as follows: the plasmid vector is used for expression, so that the loss of the plasmid is easy to cause, and the expression amount is not as stable as the genome integration; the use of inducers such as IPTG is cytotoxic and potentially risky, and is not easily removed by purification.

Disclosure of Invention

In order to solve the existing problems, the invention integrates the quorum sensing mode of the microorganisms into the genome of the Escherichia coli, expresses related genes in a 2 '-FL synthesis pathway, enables the Escherichia coli to obtain a positive feedback mechanism, and can automatically start the in vivo synthesis of the 2' -FL when the density reaches a certain threshold value.

One of the purposes of the present invention is to provide an application of microbial quorum sensing by means of genome integration to self-induced expression of related proteins in the 2' -FL synthesis pathway.

The method also provides a method for synthesizing 2 ' -FL in Escherichia coli, and the method for synthesizing 2 ' -FL is a method for synthesizing 2 ' -FL in Escherichia coli by using microbial thalli induction, and performs protein self-induced expression.

Further, in the method for synthesizing 2' -FL by using microbial quorum sensing in escherichia coli, a promoter and a structural gene corresponding to vibrio freundii quorum sensing are cloned into a plasmid to construct a self-induced gene path, wherein the self-induced gene path is luxpL-RBS-luxI-TT-luxpL-RBS-luxR-TT-luxpR-RBS-X-TT, and X is a CDS sequence of a gene to be expressed.

Further, in the method for synthesizing 2' -FL by inducing Escherichia coli with the microorganism, the Escherichia coli strain is selected from any one of BL21, K12, MG1655, TOP10 and JM 109.

Further, the method for synthesizing 2' -FL by inducing Escherichia coli with the microorganism is characterized in that JM109 is selected as a strain of Escherichia coli.

Further, the method for synthesizing 2 '-FL by inducing escherichia coli with a microorganism is characterized in that the method for synthesizing 2' -FL is performed by overexpressing one or more of gmd, fcl, manA, manB, and manC in the pathway of de novo synthesis of GDP-L-fucose; on this basis, any one or more of the genes encoding WcaA to WcaF, WcaI and WcaJ may be deleted in some cases to increase the yield.

Further, the method for synthesizing 2' -FL by inducing microorganism to escherichia coli as described above, wherein in the GDP-L-fucose salvage synthesis pathway, cytoplasmic L-fucose is converted into GDP-L-fucose by the sequential action of fucose 1-kinase and fucose-1-P guanylyltransferase, and fkp gene is additionally expressed; on this basis, in some embodiments, the genes for fucose isomerase (FucI) and fucose kinase (FucK) may be knocked out; the throughput through the salvage synthesis pathway is increased to increase yield.

Further, the method for synthesizing 2 '-FL by using the microbial quorum sensing Escherichia coli is characterized in that the method for synthesizing 2' -FL uses an alpha-1, 2-fucosyltransferase gene (futC), and the alpha-1, 2-fucosyltransferase gene is derived from any one of the following: helicobacter pylori (Helicobacter pylori), Helicobacter bilis (Helicobacter bilis), Escherichia coli (Escherichia coli), Bacteroides ovatus (Bacteroides ovatus), Prevotella ruminicola (Prevotella ruminicola), Bacteroides monoides (Bacteroides uniflora), cyanobacteria (Thermoynechococcus elongatus).

Further, the method for synthesizing 2 '-FL by quorum sensing in escherichia coli using the microbial quorum sensing gene pathway in escherichia coli JM109 using the gene editing method is characterized in that the method for synthesizing 2' -FL comprises the following steps:

constructing a luxI-luxR self-induced expression pathway;

2' -FL synthesis path construction; the first part of strategy is to replace malEFG operon after the gmd-fcl, manB and manC genes are connected in series; the expression module and the induction mode use a luxI-luxR self-induced expression pathway; the second strategy is to replace the fucok operon after the genes futC and fkp are connected in series; the expression module and the induction mode use a luxI-luxR self-induced expression pathway.

Further, the method for synthesizing 2 '-FL by using the microbial quorum sensing Escherichia coli is characterized in that one or more primers selected from SEQ ID Nos. 1 to 44 are used in the method for synthesizing 2' -FL.

Some concepts of the invention are as follows:

1. selection of E.coli strains, including but not limited to BL21, K12, MG1655, TOP10, JM 109; preferably, Escherichia coli JM109 strain is selected. Since in Escherichia coli JM109, the entire lac operon was deleted from the genome. However, JM109 strain carries F' epsilon, a modified lac operon lacIqlacZ. DELTA.M 15. The lacZ Δ M15 gene encodes an inactive β -galactosidase which retains the deletion of amino acids 11-41 in the α portion of lacZ, so JM109 lacks β -galactosidase activity.

2. Synthesis of substrate GDP-L-fucose

On the one hand, in the GDP-L-fucose de novo synthesis pathway, GDP-L-fucose is produced in high yield by overexpressing gmd, fcl, manA, manB, manC (any one or more) E.coli strains; to further convert to kossic acid by avoiding accumulation of GDP-L-fucose, various genes in several biosynthetic pathways were deleted. Including the genes encoding WcaA through WcaF, WcaI and WcaJ (any one or more).

3. Expression of fucosyltransferase

The family of Fucosyltransferases (FUT) is a class of glycosyltransferase molecules that are involved in the synthesis of cell surface glycoprotein and glycolipid sugar chains, which play a role in a variety of physiological processes. The alpha-1, 2-fucosyltransferase gene (futC) for synthesizing 2' -FL is derived from any one of the following: helicobacter pylori (Helicobacter pylori), Helicobacter bilis (Helicobacter bilis), Escherichia coli (Escherichia coli), Bacteroides ovatus (Bacteroides ovatus), Prevotella ruminicola (Prevotella ruminicola), Bacteroides monoides (Bacteroides unifomis), cyanobacteria (Thermoynechococcus elongatus), and the like. Generally, the genomic DNA of helicobacter pylori is selected, primers are designed to amplify the alpha-1, 2 or alpha-1, 3-fucosyltransferase gene FutC, and then the gene is expressed in an E.coli strain by a suitable vector.

4. Establishment of self-induction mode in quorum sensing cells

A method for self-induced expression of intracellular protein relates to the field of genetic engineering. Cloning the corresponding promoter and structural gene of Vibrio fischeri quorum sensing into plasmid to construct a self-induced gene pathway, luxpL-RBS-luxI-TT-luxpL-RBS-luxR-TT-luxpR-RBS-X-TT (X is the CDS sequence of the gene to be expressed), as shown in FIG. 1. The method can realize automatic mass induction expression of subsequent target proteins under higher thallus concentration through positive feedback of a channel without depending on an inducer, realizes separation of cell growth period and product expression time, and further improves product yield. Because no inducer is added, the cost is saved, the operation process is simplified, and on the other hand, the cytotoxicity and the purification difficulty brought by the inducer are reduced.

Drawings

FIG. 1 is a schematic diagram of the self-inducible gene pathway of the present invention;

FIG. 2 is a pTD103luxI _ sfGFP plasmid in one embodiment of the present invention;

FIG. 3 is a schematic diagram of the luxI-luxR self-inducible expression system in one embodiment of the present invention;

FIG. 4 shows the full length of the homologous replacement template;

FIG. 5 shows the integrated genomic colony PCR assay for overexpressed genes in one embodiment of the present invention;

FIG. 6 shows the total length of the homologous replacement template in an embodiment of the present invention;

FIG. 7 is a PCR identification of a colony in one embodiment of the invention;

FIG. 8 is a 2' -FL standard quality spectrum according to an embodiment of the present invention;

FIG. 9 is a mass spectrum of 2' -FL fermentation supernatant in one embodiment of the present invention.

Detailed Description

The following examples are intended to further illustrate some, but not all, preferred embodiments of the present invention. Other embodiments of the invention based on the present invention, which can be made by a person skilled in the art without inventive step, belong to the scope of protection of the present invention. The invention will be further described with reference to the accompanying drawings. The sequences involved in the present invention are all in the 5 'to 3' direction.

The experimental procedures, in which specific conditions are not indicated in the examples, are generally carried out according to the conditions conventional in the art or according to the conditions recommended by the equipment and reagent manufacturers.

Example 1 self-induced synthesis of 2-FL by integration of quorum sensing Gene into E.coli JM109 Using Gene editing method

1. The pTD103luxI _ sfGFP plasmid is derived from the adddge 48885# plasmid, the sequence and related information of which are provided by the adddge website, and the map of which is shown in FIG. 2.

2. Construction of luxI-luxR self-induced expression pathway

2.1 designing a primer, taking pTD103luxI _ sfGFP plasmid as a template, carrying out PCR amplification on a luxpL-RBS gene fragment, and carrying out gel recovery for later use;

2.2 designing a primer, taking pTD103luxI _ sfGFP plasmid as a template, carrying out PCR amplification on a luxI-rrnB T1 terminator gene fragment, and carrying out gel recovery for later use;

2.3 designing a primer, taking pTD103luxI _ sfGFP plasmid as a template, carrying out PCR amplification on a luxpL-RBS-luxR gene fragment, and carrying out gel recovery for later use;

2.4 designing a primer, taking pTD103luxI _ sfGFP plasmid as a template, amplifying rrnB T1 terminator gene fragment by PCR, and carrying out gel recovery for later use;

2.5 designing a primer, taking pTD103luxI _ sfGFP plasmid as a template, carrying out PCR amplification on a luxPR-RBS gene fragment, and carrying out gel recovery for later use;

2.6 the fragments are connected into a fragment by a Gibbson connection method, and the gel is recovered for standby after PCR amplification to obtain the fragment as shown in figure 3luxI-luxR self-induced expression system.

TABLE 1 luxI-luxR self-induced expression pathway construction primer table

Note: all primer designs contained 17-18bp overlapping bases required for Gibbson ligation.

3. 2' -FL synthetic pathway construction

3.1JM109 Strain was purchased from Takara and its genotype is E.coli JM109: recA1, endA1, gyrA96, thi-1, hsdR17(rk-mk +), e14- (mcrA-), supE44, relA1, Delta (lac-proAB)/F' [ traD36, proAB +, lacIq, lacZ Delta M15]

3.2 inoculating JM109 strain to 5ml LB non-resistant liquid medium, culturing for 12-16 hours, extracting genome and storing at-20 deg.C;

3.3 this example first part strategy is to replace the malEFG operon with the gmd-fcl, manB, manC genes in tandem; the expression module and the induction mode use a luxI-luxR self-induced expression pathway.

(1) Designing a primer, and carrying out PCR amplification by taking JM109 genome as a template to obtain gmd-fcl, manB and manC gene fragments and carrying out gel recovery for later use;

TABLE 2 overexpression Gene amplification PCR primers

(2) Determining the position of the replacement and selecting a proper N20 sequence according to the sequence information of the malEFG operon; primers were designed, and after linear plasmids were obtained by PCR using pTargetF plasmid (derived from Addgene #62226) as a template, DH5a competent cells were transformed by Gibbson ligation and plated in an incubator at 37 ℃ overnight. Selecting monoclonal shake bacteria and sequencing the single shake bacteria, successfully sequencing the plasmid replacing the N20 sequence to be a positive clone, selecting the positive clone shake bacteria to culture and extracting the plasmid to obtain malEFG-pTargetF1

And malEFG-pTargetF2 plasmid.

TABLE 3 pTargetF-N20 plasmid construction primer Table

(3) Based on the adjacent sequences upstream and downstream of the malEFG operon, a homologous template of an appropriate size is designed, and a primer is designed. Taking JM109 genome as a template, carrying out PCR amplification on upstream and downstream homology arms, and recovering for later use.

TABLE 4 homologous arm amplification PCR primers

(4) And performing Gibbson connection on the upstream homology arm, the luxI-luxR self-induction system, the gmd-fcl, the manB, the manC, the rrnB T1 terminator and the downstream homology arm, performing PCR amplification to obtain a homology replacement template, and recovering the glue for later use. As shown in fig. 4. (5) The full length of malEFG-pTargetF1 plasmid, malEFG-pTargetF2 plasmid and homologous template were simultaneously electroporated into JM109 E.coli previously transformed with pCas plasmid (derived from addge #62225), and cultured overnight at 30 ℃ after plating kanamycin and spectinomycin double resistant plates. And selecting the monoclone the next day, carrying out colony PCR identification, and identifying the positive strain as a successfully replaced strain.

TABLE 5 colony PCR identification primers

(6) Elimination of pCas and pTargetF

The correctly identified clones containing pCas and Lon-sgRNA-pTargetF were inoculated into 2mL of liquid LB medium containing kanamycin (50. mu.g/mL) and IPTG (isopropyl-. beta. -D-thiogalactopyranoside, 0.5mmol/L), cultured for 8 to 16 hours and plated onto LB agar containing kanamycin (50. mu.g/mL). Then, it was confirmed that pTargetF had been eliminated by the sensitivity of the clone to spectinomycin (50. mu.g/mL). The clones were cultured overnight at 43 ℃ to eliminate the pCas plasmid. The strain JM109-001 is obtained and stored for further use.

3.4 the second partial strategy of this example is to replace the fucok operon after the futC, fkp genes are concatenated; the expression module and the induction mode use a luxI-luxR self-induced expression pathway.

(1) Synthetic futC (from helicobacter pylori), and cloned into pCOLADuet-1 plasmid between BamH I and NotI cleavage sites; designing a primer, taking pCOLADuet-1 plasmid as a template, carrying out PCR amplification to obtain a futC gene fragment, and carrying out gel recovery for later use.

(2) Fkp gene (from Bacteroides fragilis) was synthesized and cloned between Bgl II and KpnI cleavage sites of pCOLADuet-1 plasmid; designing a primer, taking pCOLADuet-1 plasmid as a template, carrying out PCR amplification to obtain fkp gene fragments, and carrying out gel recovery for later use.

TABLE 6 exogenous Gene amplification PCR primers

(3) Determining the position of the substitution and selecting a suitable N20 sequence according to the sequence information of the fucoK operon; primers were designed, and after linear plasmids were obtained by PCR using pTargetF plasmid (derived from Addgene #62226) as a template, DH5a competent cells were transformed by Gibbson ligation and plated in an incubator at 37 ℃ overnight. Selecting monoclonal shake bacteria and sequencing the monoclonal shake bacteria, successfully sequencing the plasmid replacing the N20 sequence to be a positive clone, selecting the positive clone to shake bacteria and culturing the positive clone and extracting the plasmid to obtain FucIK-pTargetF1

And the FucIK-pTargetF2 plasmid.

TABLE 7 pTargetF-N20 plasmid construction primer Table

(3) From the adjacent sequences upstream and downstream of the fucok operon, a homologous template of an appropriate size was designed, and primers were designed. Taking JM109 genome as a template, carrying out PCR amplification on upstream and downstream homology arms, and recovering for later use.

TABLE 8 homologous arm amplification PCR primers

(4) And performing PCR amplification on the upstream homology arm, the luxI-luxR self-induction system, the futC, the fkp, the rrnB T1 terminator and the downstream homology arm after Gibbson connection to obtain a homology replacement template, and recovering the glue for later use. FIG. 6, below, replaces the template homologously for the full length.

(5) The full length of FucIK-pTargetF1 plasmid, FucIK-pTargetF2 plasmid and homologous template were simultaneously electroporated into JM109-001 E.coli previously transformed with pCas plasmid (derived from addge #62225), and cultured overnight at 30 ℃ after plating kanamycin and spectinomycin double resistant plates. The single clone is selected the next day, colony PCR identification is carried out, and the identified positive strain is a successfully replaced strain and is named as JM 109-002.

TABLE 9 colony PCR identification primers

As identified by colony PCR in figure 7.

(6) Elimination of pCas and pTargetF

The correctly identified clones containing pCas and Lon-sgRNA-pTargetF were inoculated into 2mL of liquid LB medium containing kanamycin (50. mu.g/mL) and IPTG (isopropyl-. beta. -D-thiogalactopyranoside, 0.5mmol/L), cultured for 8 to 16 hours and plated onto LB agar containing kanamycin (50. mu.g/mL). Then, it was confirmed that pTargetF had been eliminated by the sensitivity of the clone to spectinomycin (50. mu.g/mL). The clones were cultured overnight at 43 ℃ to eliminate the pCas plasmid. The obtained strain JM109-002 is preserved for later use.

3.5 fermentation of Escherichia coli JM109-002 and identification of 2' -FL

(1) Preparing 200ml LB liquid culture medium, glycerol (working concentration is 10g/L, mother liquor is prepared to be 1g/ml), L-fucose (working concentration is 2g/L, mother liquor is prepared to be 0.2g/ml), lactose (working concentration is 10g/L, mother liquor is prepared to be 1g/ml), sterilizing for later use;

(2) preculture

Inoculating JM109-002 strain in 100ml LB culture medium, shaking at 30 deg.C and 220rpm, and culturing for 16 hr;

(3) formal fermentation

Weighing the pre-cultured thallus, inoculating the pre-cultured thallus into 200ml of LB culture medium according to the dry weight of 3.6g/L cells, adding 10g/L of glycerol, 10g/L of lactose and 2g/L of L-fucose, and carrying out shake flask fermentation at 25 ℃ and 300rpm for 72 hours; each 24 hours of fermentation culture was supplemented with 6g/L of glycerol, 6g/L of lactose, 2g/L of L-fucose until the end of fermentation.

(4) Harvesting the supernatant

Centrifuging the fermentation liquor, collecting the culture supernatant, filtering with a sterile filter membrane, sampling, and detecting whether the product 2' -FL is synthesized by mass spectrometry. The results are shown in FIGS. 8 and 9 below, and the results were obtained for the synthesis of 2' -FL.

The foregoing examples are intended to further illustrate some preferred embodiments of the invention, not all embodiments. Other embodiments of the invention based on the present invention, which can be made by a person skilled in the art without inventive step, belong to the scope of protection of the present invention.

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cctgccttag accattctga 20

<210> 27

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 27

caccttcatg gatatcgaga 20

<210> 28

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 28

ctactcagga gagcgttcac 20

<210> 29

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 29

aagaggagaa aggtaccatg gcttttaagg tggtgca 37

<210> 30

<211> 48

<212> DNA

<213> Artificial Sequence

<400> 30

acctttctcc tctttaatga attcatttaa gcgttatact tttgggat 48

<210> 31

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 31

ttaaagagga gaaaggtacc atgcaaaaac tactatcttt ac 42

<210> 32

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 32

atgcctctag cacgcgttta tgatcgtgat acttgga 37

<210> 33

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 33

gacgaccgtc aataaccggg cgg 23

<210> 34

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 34

gaccgtcaat aaccggggtt ttagagctag aaatagcaag tta 43

<210> 35

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 35

cccggttatt gacggtcgtc actagtatta tacctaggac tgagctagct 50

<210> 36

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 36

ctgataatga atctgtgcgc ggg 23

<210> 37

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 37

ataatgaatc tgtgcgcgtt ttagagctag aaatagcaag tta 43

<210> 38

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 38

gcgcacagat tcattatcag actagtatta tacctaggac tgagctagct 50

<210> 39

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 39

attctgctat gtcggcgcac 20

<210> 40

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 40

tgtaggatcg tacaggttac agattcctca ctttatt 37

<210> 41

<211> 37

<212> DNA

<213> Artificial Sequence

<400> 41

gccgtcttca cctcgagaat gctgaaaaca atttcgc 37

<210> 42

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 42

agcagcaggt cgactatcgc 20

<210> 43

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 43

atcacagtga cgccaaacaa 20

<210> 44

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 44

tgtaggtaat aaacgccagc 20

Claims

1. Use of a microbial quorum sensing agent, wherein the microbial quorum sensing agent is used for self-inducible expression of a protein related to the 2' -FL synthesis pathway by means of genomic integration.

2. A method for synthesizing 2 ' -FL in Escherichia coli, characterized in that the method for synthesizing 2 ' -FL is a method for synthesizing 2 ' -FL in Escherichia coli by using microbial quorum sensing, and protein self-induced expression is carried out.

3. The method for synthesizing 2 '-FL by using microbial quorum sensing in Escherichia coli according to claim 2, wherein the method for synthesizing 2' -FL comprises cloning promoters and structural genes corresponding to Vibrio fischeri quorum sensing into plasmids to construct a self-inducible gene pathway, which is luxpL-RBS-luxI-TT-luxpL-RBS-luxR-TT-luxpR-RBS-X-TT, wherein X is a CDS sequence of a gene to be expressed.

4. The method for synthesizing 2' -FL by using microbial quorum sensing Escherichia coli according to claim 2, wherein the strain of Escherichia coli is selected from any one of BL21, K12, MG1655, TOP10, and JM 109.

5. The method of claim 4, wherein the strain of Escherichia coli is JM 109.

6. The method for synthesizing 2 '-FL in Escherichia coli by using microbial quorum sensing according to claim 2, wherein the method for synthesizing 2' -FL is performed by overexpressing any one or more of gmd, fcl, manA, manB and manC in the pathway of GDP-L-fucose de novo synthesis.

7. The method for synthesizing 2 '-FL in Escherichia coli by using microbial quorum sensing according to claim 6, wherein the method for synthesizing 2' -FL converts cytoplasmic L-fucose to GDP-L-fucose by sequential actions of fucose 1-kinase and fucose-1-P guanylyltransferase in a GDP-L-fucose salvage synthesis pathway, and increases the flux through the salvage synthesis pathway by additionally expressing fkp gene.

8. The method for synthesizing 2 '-FL in Escherichia coli by using microbial quorum sensing according to claim 6, wherein said method for synthesizing 2' -FL uses an α -1, 2-fucosyltransferase gene (futC), said α -1, 2-fucosyltransferase gene being derived from any one of the following: helicobacter pylori (Helicobacter pylori), Helicobacter bilis (Helicobacter bilis), Escherichia coli (Escherichia coli), Bacteroides ovatus (Bacteroides ovatus), Prevotella ruminicola (Prevotella ruminicola), Bacteroides monoides (Bacteroides uniflora), cyanobacteria (Thermoynechococcus elongatus).

9. The method for synthesizing 2 '-FL by sensing Escherichia coli with a microorganism according to claim 2, wherein the method for synthesizing 2' -FL comprises the following steps:

constructing a luxI-luxR self-induced expression pathway;

10. The method for synthesizing 2 '-FL by using microbial quorum sensing Escherichia coli according to claim 9, wherein one or more primers selected from SEQ ID Nos. 1 to 44 are used in the method for synthesizing 2' -FL.