Method for producing 2' -fucosyllactose using fucosyltransferase derived from Agrobacterium choreoides
阅读说明：本技术 利用源自舞蹈拟土地杆菌的岩藻糖基转移酶的2’-岩藻糖基乳糖的生产方法 (Method for producing 2' -fucosyllactose using fucosyltransferase derived from Agrobacterium choreoides ) 是由 申哲寿 尹钟远 宋永厦 柳英善 金甫美 金镇景 李佳恩 于 2019-02-14 设计创作，主要内容包括：本发明涉及一种从插入源自舞蹈拟土地杆菌(Pseudopedobater saltans)的岩藻糖基转移酶(fucosyltransferase)的重组棒状杆菌属(Corynebacterium SP.)微生物中生产2’-岩藻糖基乳糖的方法,本发明的插入源自舞蹈拟土地杆菌(Pseudopedobater saltans)的岩藻糖基转移酶(fucosyltransferase)的重组棒状杆菌属(Corynebacterium SP.)微生物比常规大肠杆菌更安全,同时可以高浓度、高产率、高生产率生产2’-岩藻糖基乳糖。(The present invention relates to a method for producing 2'-fucosyllactose from a microorganism of the genus Corynebacterium (Corynebacterium SP) having an inserted fucosyltransferase (fucosyltransferase) derived from Pseudoterreus dancaci (Pseudomonas saltans), which is safer than conventional Escherichia coli and can produce 2' -fucosyllactose in high concentration, high yield and high productivity.)
1. A recombinant microorganism belonging to the genus Corynebacterium (SP), characterized in that,
which is transformed to express an alpha-1, 2-fucosyltransferase (alpha-1, 2-fucosyltransferase) derived from a strain of Agrobacterium choreoides (Pseudomonas saltans) having an amino acid sequence corresponding to SEQ ID NO. 5,
converted to express GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase),
converting into GDP-L-fucose synthase (GDP-L-fucose synthase),
conversion to express lactose permease (lactose permase),
and retains phosphomannomutase (phosphomannomutase) and GTP-mannose-1-phosphate guanylyltransferase (GTP-mannose-1-phosphate guanylyltransferase).
2. The recombinant microorganism of the genus Corynebacterium (Corynebacterium SP) according to claim 1,
the recombinant Corynebacterium SP microorganism is any one selected from the group consisting of Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium ammoniagenes (Corynebacterium ammoniagenes), and Corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes).
3. The recombinant microorganism of the genus Corynebacterium (Corynebacterium SP) according to claim 1,
the above-mentioned α -1,2-fucosyltransferase (α -1,2-fucosyltransferase) having an amino acid sequence corresponding to sequence No. 5 is encrypted with the nucleic acid sequence shown in sequence No. 4.
4. The recombinant microorganism of the genus Corynebacterium (Corynebacterium SP) according to claim 1,
the recombinant Corynebacterium SP is transformed to overexpress phosphomannose mutase,
the conversion is carried out to over-express GTP-mannose-1-phosphate guanine transferase (GTP-mannose-1-phosphate guanylyltransferase).
5. A production method of 2' -fucosyllactose is characterized in that,
culturing the recombinant microorganism of the genus Corynebacterium (Corynebacterium SP) according to claim 1 in a medium supplemented with lactose.
6. The method for producing 2' -fucosyllactose according to claim 4,
the medium further comprises glucose.
Technical Field
The present invention relates to a method for producing 2'-fucosyllactose (2' -fucosyllactose,2'-FL), and more particularly, to a method for producing 2' -fucosyllactose from a recombinant Corynebacterium (Corynebacterium SP.) microorganism into which fucosyltransferase (fucosyltransferase) derived from geobacter choreoides (pseudopodobacter saltans) is inserted.
Background
Compared with milk of other mammals, more than 200 kinds of oligosaccharides (HMOs) having unique structures are present in human breast milk at a considerably high concentration (5 to 15 g/L). HMOs consist of D-glucose (Glc), D-galactose, N-acetylglucosamine (GlcNAc), L-fucose (Fuc) and sialic acid (sialc acid) [ Sia; n-acetyl neuroaminic acid (Neu5Ac) ].
Since the structure of HMOs is very diverse and complex, about 200 isomers with other residues and glycosylation bonds may exist at mutually different degrees of polymerization (DP 3-20). However, despite their complex structure, HMOs still exhibit several common structures, with most HMOs having a lactose (Gal β 1-4Glc) residue at the reducing end. The Gal of lactose can be in the form of 3-sialyllactose (3-sialyllactose) or 6-sialyllactose (6-sialyllactose) in the form of the α - (2,3) -and α - (2,6) -linkages, respectively, or in the form of 2-fucosyllactose (2' -fucosylase, 2' -FL) or 3-fucosyllactose (3' -fucosylase, 3-FL) in the form of the α - (1,2) -and α - (1,3) -linkages, respectively, fucosylation (fucosylation).
137 including the three oligosaccharides with the highest content in breast milk were fucosylated at a rate of about 77%, while the remaining oligosaccharides were mostly salified (39), about 28%. Among them, 2-fucosyllactose and 3-fucosyllactose are the main HMOs providing various biological activities that contribute to prebiotic (prebiotic) effects of intestinal lactic acid bacteria growth, preventing infection by pathogenic bacteria, regulating the immune system, and having positive effects on the development and health of infants such as brain development, and thus it is very important that experts emphasize breast feeding during infancy.
However, it is known that approximately 20% of women cannot normally synthesize fucosyloligosaccharides (fucosyloligosaccharides) in vivo due to a mutation in fucosyltransferase (fucosyltransferase) that synthesizes them. Therefore, the industrial production of fucosyllactose is actually required.
On the other hand, as a method for producing fucosyllactose, there are a method of directly extracting from breast milk and a method of synthesizing by a chemical or enzymatic method. The direct extraction method has problems of limited supply and demand of breast milk and low productivity, and the chemical synthesis method has problems of expensive substrate, low isomer selectivity (stereo-selectivity) and production yield, and the use of toxic organic solvents. Further, the enzymatic synthesis method has problems that GDP-L-fucose used as a fucose donor (dornor) is very expensive and that fucosyltransferase is expensive to be purified.
Due to the above problems, direct extraction, chemical or enzymatic production methods are difficult to apply to mass production of fucosyllactose, and as a solution to this problem, a solution for producing 2' -fucosyllactose using microorganisms has been proposed. The prior art for producing 2' -fucosyllactose using microorganisms is mostly a technique using recombinant E.coli production. However, most of E.coli used in the experiments are not actually pathogenic bacteria, but are widely recognized as harmful bacteria by consumers.
In addition, since the cell membrane components of Escherichia coli may act as endotoxins, the separation and purification cost is high when 2' -fucosyllactose is produced. Therefore, it has been difficult to use Escherichia coli as a host cell for fucosyllactose, which is a raw material for producing foods and pharmaceuticals.
Disclosure of Invention
Technical subject
The present invention aims to provide a method for producing 2' -fucosyllactose using a recombinant Corynebacterium (Corynebacterium SP.) microorganism into which fucosyltransferase (fucosyltransferase) derived from agrobacterium choreoides (pseudopodobacter saltans) is inserted as a host cell for fucosyllactose, which is a raw material for producing foods and pharmaceuticals.
Technical subject
The present invention provides a recombinant microorganism belonging to the genus Corynebacterium SP, which is transformed to express an alpha-1, 2-fucosyltransferase (alpha-1, 2-fucosyltransferase) derived from Geobacillus choracearum (Pseudomonas SP) and having an amino acid sequence corresponding to SEQ ID NO. 5, to express GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase), to express GDP-L-fucose synthase (GDP-L-fucose synthase), and to express lactose permease (lactose permease), and retains phosphomannomutase (phosphomannomutase) and GTP-mannose-1-phosphate guanylyltransferase (GTP-mannose-1-phosphate guanylyltransferase).
Preferably, the recombinant Corynebacterium SP microorganism of the present invention is any one selected from the group consisting of Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium ammoniagenes (Corynebacterium ammoniagenes), and Corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes).
In the recombinant microorganism of the present invention, preferably, the α -1,2-fucosyltransferase (α -1,2-fucosyltransferase) having the amino acid sequence corresponding to SEQ ID NO. 5 is encrypted with the nucleic acid sequence of SEQ ID NO. 4.
In the recombinant coryneform bacterium (Corynebacterium SP) of the present invention, preferably, the above-mentioned recombinant coryneform bacterium (Corynebacterium SP) is transformed to overexpress phosphomannose mutase (phosphomannose) and to overexpress GTP-mannose-1-phosphate guanine transferase (GTP-mannose-1-phosphate guanylyltransferase).
The present invention also provides a method for producing 2' -fucosyllactose, comprising culturing the recombinant microorganism belonging to the genus Corynebacterium (Corynebacterium SP.) of the present invention in a medium supplemented with lactose.
In the method for producing 2' -fucosyllactose according to the present invention, the culture medium preferably further contains glucose.
Effects of the invention
The recombinant coryneform bacterium (Corynebacterium SP) microorganism of the present invention, into which fucosyltransferase (fucosyltransferase) derived from Pseudocerus choracearum (Pseudomonas salts) is inserted, is safer than conventional Escherichia coli, and can produce 2' -fucosyllactose in high concentration, high yield and high productivity.
Drawings
FIG. 1 is a schematic representation of pFGW (Ps) plasmid.
FIG. 2 is a schematic representation of pXIL plasmid.
FIG. 3 is a schematic representation of pFGW (Hp) plasmid.
FIG. 4 is a result of culturing a recombinant strain inserted with fucosyltransferase (fucosylransferase) derived from Helicobacter pylori (Helicobacter pylori) (●: dried cell weight, ■: lactose,: glucose,. diamond-solid.: 2' -FL,. lactate,. O.: acetate).
FIG. 5 is a result of culturing a recombinant strain in which fucosyltransferase derived from Agrobacterium choreofaciens (Pedobacter saltans) was inserted (●: dry cell weight, ■: lactose,. t.t.. glucose,. t. 2' -FL.,. lactate. O. acetate.).
Detailed Description
The present invention provides a recombinant microorganism belonging to the genus Corynebacterium SP, which is transformed to express an alpha-1, 2-fucosyltransferase (alpha-1, 2-fucosyltransferase) derived from Geobacillus choracearum (Pseudomonas SP) and having an amino acid sequence corresponding to SEQ ID NO. 5, to express GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase), to express GDP-L-fucose synthase (GDP-L-fucose synthase), and to express lactose permease (lactose permease), and retains phosphomannomutase (phosphomannomutase) and GTP-mannose-1-phosphate guanylyltransferase (GTP-mannose-1-phosphate guanyltransferase).
Preferably, the recombinant Corynebacterium SP microorganism of the present invention is any one selected from the group consisting of Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium ammoniagenes (Corynebacterium ammoniagenes), and Corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes).
On the other hand, Brevibacterium flavum (Brevibacterium flavum) is currently classified as Corynebacterium glutamicum (Corynebacterium glutamicum), and thus Brevibacterium flavum (Brevibacterium flavum) strains are also included in the scope of the present invention.
Also, according to the UniProt database, Brevibacterium saccharolyticum (Brevibacterium saccharolyticum) is a synonym OF Corynebacterium glutamicum (Corynebacterium glutamicum), AND Brevibacterium lactofermentum (Brevibacterium lactofermentum) is also named as a nickname OF Corynebacterium glutamicum (Corynebacterium glutamicum), AND thus Brevibacterium saccharolyticum (Brevibacterium saccharolyticum) AND Brevibacterium lactofermentum (Brevibacterium lactofermentum) are also included in the scope OF the present invention (W.LIEBL et al, INTERNATIONAL JUNGUANAL OF STEMATIBCA CTIOLOGY, Apr.1991, p.255-260; LOAR GEEGLING et al, JOURNAUS OF BIOSCENCE AND BINGING OEL.92, JOUR 3,201-213.2001; Haynergs et al, J. 329, FEYNER OF BIOSCENCE AND MICROBINGING OEMS 334).
The present inventors previously obtained a patent of a production method of 2' -fucosyllactose using corynebacteria through korean granted patent No. 10-17312630000 (2017.04.24). In the present invention, fucosyllactose (fucosyltransferase) derived from terribacterium choriformis (pseudoperobacterium villans) is inserted into corynebacteria, so that 2'-fucosyllactose can be obtained in a significantly larger amount than 2' -fucosyllactose conventionally produced by inserting fucosyltransferase (fucosyltransferase) derived from helicobacter pylori (helicobacter pylori) into corynebacteria.
On the other hand, unlike conventionally used escherichia coli, Corynebacterium glutamicum (Corynebacterium glutamicum) or Corynebacterium ammoniagenes (Corynebacterium ammoniagenes) are not only strains recognized as gras (genetically recovered as safe), but also strains that do not produce endotoxin and are widely used in industrial production of amino acids and nucleic acids as food additives. Thus, Corynebacterium glutamicum (Corynebacterium glutamicum) or Corynebacterium ammoniagenes (Corynebacterium ammoniagenes) can be said to be strains suitable as raw materials for the production of foods and pharmaceuticals, and have the advantage that consumer concerns about safety can be eliminated.
On the other hand, since strains of Escherichia coli and a microorganism of Corynebacterium genus (Corynebacterium SP) including Corynebacterium glutamicum (Corynebacterium glutamicum), Corynebacterium ammoniagenes (Corynebacterium ammoniagenes), and Corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes) have different genetic characteristics from each other, it is necessary to use a strategy different from the strategy applied to Escherichia coli. In order to produce 2' -fucosyllactose, Escherichia coli and microorganisms of the genus Corynebacterium (Corynebacterium SP.) are required to introduce exogenous alpha-1, 2-fucosyltransferase (alpha-1, 2-fucosyltransferase) basically, but microorganisms of the genus Corynebacterium (Corynebacterium SP.) are required to further introduce GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4, 6-degydrase, Gmd), GDP-L-fucose synthase (GDP-L-fucose synthase, Wcag), lactose permease (lactose, LacY) in addition to the microorganisms.
In this case, in the recombinant microorganism of the present invention belonging to Corynebacterium SP, it is preferable that the α -1,2-fucosyltransferase (α -1,2-fucosyltransferase) having the amino acid sequence corresponding to SEQ ID NO. 5 is encrypted with the nucleic acid sequence described in SEQ ID NO. 4, and as the encrypted GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase, Gmd), GDP-L-fucose synthase (GDP-L-fucose synthase, GDP-4-keto-6-deoxymannase-3, 5-endogerase-4-reductase, Wcag) and lactose permease (lactonase, LacY) genes, a common gene may be used, and a gene derived from escherichia coli may be preferably used. However, lactose permease (LacY) is an enzyme involved in the transport of lactose existing outside the strain to the inside of the strain, and in the present invention, the Lac operon is introduced for the purpose of introducing lactose, so that only the LacY gene is introduced, and there is no need to introduce the lacA gene.
On the other hand, the recombinant microorganism of Corynebacterium SP may retain the genes encoding phosphomannomutase (ManB), GTP-mannose-1-phosphate guanylyltransferase (GTP-mannose-1-phosphate guanylyltransferase, ManC) and express them, and thus it is not necessary to overexpress the enzymes, but it is necessary to overexpress the enzymes for mass production. Therefore, in the recombinant Corynebacterium SP microorganism of the present invention, preferably, the recombinant Corynebacterium SP microorganism is transformed to overexpress phosphomannose mutase (phosphomannose) and to overexpress GTP-mannose-1-phosphate guanine transferase (GTP-mannose-1-phosphoguanylyltransferase).
The present invention also provides a method for producing 2' -fucosyllactose, comprising culturing the recombinant microorganism belonging to the genus Corynebacterium (Corynebacterium SP.) of the present invention in a medium supplemented with lactose. According to the experiments of the present invention described below, 2' -fucosyllactose can be produced from recombinant Corynebacterium SP microorganisms at a high concentration, a high yield, and a high productivity, as compared to the use of existing strains.
On the other hand, in the method for producing 2' -fucosyllactose of the present invention, it is preferable that the culture medium further contains glucose at a high concentration. By adding glucose to the medium, the growth of the strain becomes active, so that 2' -fucosyllactose can be produced with higher productivity.
On the other hand, it is preferable that the method for producing 2' -fucosyllactose of the present invention is fed-batch culture with additional supply of glucose or lactose. This is because continuous supply of glucose or lactose by fed-batch culture further increases the growth of cells and produces fucosyllactose with high purity, high yield and high productivity. As for the specific details of the fed-batch culture, those known in the art can be used, and therefore, the description thereof is omitted.
The present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following examples, and may include modifications of the technical ideas equivalent thereto.
On the other hand, although the effect is confirmed below by using only Corynebacterium glutamicum (Corynebacterium glutamicum) as host cells, the same effect can be expected since Corynebacterium glutamicum (Corynebacterium glutamicum) and Corynebacterium ammoniagenes (Corynebacterium ammoniagenes), Corynebacterium thermoaminogenes (Corynebacterium thermoaminogenes), Brevibacterium flavum (Brevibacterium flavum), and Brevibacterium lactofermentum (Brevibacterium lactofermentum) can be applied to the transformation system in the same manner.
Production example 1: preparation of recombinant plasmid
For the preparation of plasmids and the production of 2'-fucosyllactose (2' -FL), Escherichia coli (Escherichia coli) K-12MG1655 and Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 were used, respectively.
To construct pFGW (Ps) plasmids, a gmd-wcaG gene cluster (cluster) was amplified from genomic (genomic) DNA of Escherichia coli, K-12MG1655, by a PCR reaction using two DNA primers (primer) GW-F and GW-R, and a pSod-Gmd-Wcag DNA fragment was synthesized by an overlap (overlap) PCR reaction using two DNA primers Sod-F and GW-R, after amplifying the promoter of the Sod gene from genomic DNA of Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13032 by a PCR reaction using two DNA primers Sod-F and Sod-R.
After amplifying the transcription termination sequence from pXMJ19 plasmid (plasmid) by PCR reaction using two DNA primers, Ter-F and Ter-R, pSod-Gmd-Wcag-Ter sequence was synthesized by PCR reaction using two DNA primers, Sod-F and Ter-R, using pSod-Gmd-Wcag and the transcription termination sequence synthesized above as templates, and then inserted into pCES208 plasmid cleaved with restriction enzyme BamHI, thereby constructing pGW plasmid.
And, after amplifying Tuf gene promoter from genomic DNA of Corynebacterium glutamicum ATCC13032 by PCR reaction using two DNA primers Tuf-F1 and Tuf-R1 and alpha-1, 2-fucosyltransferase (alpha-1, 2-fucosyltransferase) derived from synthetic Agrobacterium choreoides (Pseudomonas saltana) DSM 12145 by PCR reaction using two DNA primers FT (Ps) -F and FT (Ps) -R, pTuf-FT (Ps) DNA fragment was synthesized by overlapping (overlapping) PCR reaction using two primers Tuf-F and FT (Ps) -R. The pGW plasmid constructed as described above was treated with restriction enzyme Not I to insert pTuf-FT (Ps) into the plasmid, thereby constructing pFGW (Ps) plasmid.
On the other hand, in order to construct pXIL plasmid, lacY gene was amplified from the genomic DNA of Escherichia coli K-12MG1655 by PCR reaction using two DNA primers ilvC-lacY-F and lacY pX-R, and after the promoter of ilvC gene was amplified from the genomic DNA of Corynebacterium glutamicum ATCC13032 by PCR reaction using two DNA primers pX-ilvC-F and ilvC-lacY-R, a pilvC-lacY DNA fragment was synthesized by overlap (overlap) PCR reaction using two DNA primers pX-ilvC-F and lacY pX-R, and then inserted into pX plasmid (pXMJ19) treated with restriction enzymes Not I and EcoRI, thereby constructing pXIL plasmid.
On the other hand, for the construction of pFGW (Hp) vector, Tuf gene promoter was amplified from the genomic DNA of Corynebacterium glutamicum ATCC13032 by PCR reaction using two DNA primers Tuf-F1 and Tuf-R2 using the pGW plasmid constructed as described above, and pTuf-FT (FT) DNA fragment was synthesized by overlap PCR reaction using two primers Tuf-F1 and FT (Hp) -Ps after alpha-1, 2-fucosyltransferase (alpha-1, 2-fucosyltransferase) derived from Helicobacter pylori (Helicobacter pylori) ATCC 700392 synthesized by codon optimization (codon optimization) of Corynebacterium glutamicum was amplified by PCR reaction using two DNA primers FT (Hp) -F and FT (Hp) -R. The plasmid pFGW (Hp) was constructed by inserting pTuf-FT (Hp) into the pGW plasmid constructed as described above by treating it with a restriction enzyme Not I.
The strains (strain), primers (primer), plasmids (plasmid), nucleic acids, and amino acid sequences used in the present production examples are shown in tables 1 to 4 below.
[ Table 1]
Bacterial strains
E.Coli K-12 MG1655
F-,lambda-,rph-1
C.glutamicum
Wild-type strain,ATCC13032
[ Table 2]
Nucleic acid and amino acid sequences
gmd nucleic acid sequence
Sequence No. 1
wcAG nucleic acid sequence
Sequence number 2
lacY nucleic acid sequence
Sequence No.3
FT (Ps) nucleic acid sequence
Sequence number 4
FT (Ps) amino acid sequence
Sequence number 5
FT (Hp) nucleic acid sequence
Sequence number 6
FT (Hp) amino acid sequence
Sequence number 7
[ Table 3]
Primer and method for producing the same
Primer and method for producing the same
Sequence (5'→ 3')
pX-ilvC-F
GTCATATGATGGTCGCGGATCCGAATTCCCAGGCAAGCTCCGC
ilvC-lacY-R
GTTTTTTAAATAGTACATAATCTCGCCTTTCGTAAAAATTTTGGT
ilvC-lacY-F
TTACGAAAGGCGAGATTATGTACTATTTAAAAAACACAAACTTTTGGATGTTCGG
lacY pX-R
GCCTTTCGTTTTATTTGCTCGAGTGCGGCCGCTTAAGCGACTTCATTCACCTGACGAC
Tuf-F1
TGGAGCTCCACCGCGGTGGCTGGCCGTTACCCTGCGAA
Tuf-R1
CAAATATCATTGTATGTCCTCCTGGACTTCG
FT(ps)-F
AGGACATACAATGATATTTGTAACCGGATATG
FT(ps)-R
CGCTTCACTAGTTCTAGAGCTTAAATAATGTGTCGAAACAGATTC
Sod-F
GCTCTAGAACTAGTGAAGCGCCTCATCAGCG
Sod-R
TACACCGGTGATGAGAGCGACTTTTGACATGGTAAAAAATCCTTTCGTAGGTTTCCGCAC
GW-F
ATGTCAAAAGTCGCTCTCATCACCGGTGTA
GW-R
CAAGCTGAATTCTTACCCCCGAAAGCGGTC
ter-F
GACCGCTTTCGGGGGTAAGAATTCAGCTTG
ter-R
GGTATCGATAAGCTTGATATCGAATTCCTGCAGCCCGGGGAAAAGGCCATCCGTCAGGAT
Tuf-R2
TGAAAGCCATTGTATGTCCTCCTGGACTTCGT
FT(Hp)-F
GGACATACAATGGCTTTCAAGGTGGTCCAAAT
FT(Hp)-R
GCTTCACTAGTTCTAGAGCTTAAGCATTGTATTTCTGGCTCTTCACTTCG
[ Table 4]
Plasmids
Example 1: cultivation of recombinant Strain having fucosyltransferase (fucosyltransferase) inserted thereinto derived from Pseudocerobacter choracearum
In an experiment containing 5mL of BHI (brain Heart infusion) medium containing 25. mu.g/mL of kanamycin (kanamycin) and 5. mu.g/mL of chloramphenicol, Corynebacterium glutamicum (C.glutamicum) ATCC13032, into which pFGW (Ps, FIG. 1) and pXIL (FIG. 2) were inserted, was inoculated at 30 ℃ and 250rpm for 12 hours. FIG. 1 is a schematic representation of pFGW (Ps) plasmid, and FIG. 2 is a schematic representation of pXIL plasmid.
Batch culture utilized a minimum medium ((NH) containing 50 mL)4)2SO4 20g/L，Urea 5g/L，KH2 PO41g/L，K2HPO4 1g/L，MgSO4·7H2O 0.25g/L，MOPS 42g/L，CaCl2 10mg/L，Biotin 0.2mg/L，Protocatechuic acid 30mg/L，FeSO4·7H2O 10mg/L，MnSO4·H2O 10mg/L，ZnSO4·7H2O 1mg/L，CuSO4 0.2mg/L，NiCl2·6H2O0.02 mg/L, Glucose 40g/L, Lactose 10g/L, pH7.0) at 30 ℃ for 90 hours at 250 rpm.
Comparative example 1: cultivation of recombinant Strain having fucosyltransferase (fucosyltransferase) inserted thereinto derived from Helicobacter pylori (Helicobacter pylori) ]
The same culture method as in example 1 was used except that pFGW (Ps, fig. 1) was changed to pFGW (Hp, fig. 3). FIG. 3 is a schematic representation of pFGW (Hp) plasmid.
[ Experimental example 1: example 1 and comparative example 1 production comparison of 2' -FL by recombinant Strain
The yields of 2' -FL in the recombinant strains produced in example 1 and comparative example 1 were compared (fig. 4, fig. 5, table 5). FIG. 4 is the result of culturing a recombinant strain into which fucosyltransferase derived from Helicobacter pylori (Helicobacter pylori) is inserted, and FIG. 5 is the result of culturing a recombinant strain into which fucosyltransferase derived from Pseudomonas aeruginosa is inserted (●: dry cell weight, ■: lactose,: glucose,: diamond-solid: 2' -FL,: lactate,. smallcircle: acetate).
[ Table 5]
As a result of the experiment, it was confirmed that the 2' -fucosyllactose productivity of the recombinant strain having fucosyltransferase (fucosyllactose) derived from Agrobacterium choreoides was about 2 times higher than that of the recombinant strain having fucosyltransferase (fucosyllactose) derived from Helicobacter pylori (Helicobacter pylori).
<110> advanced protein technology Co
<120> production method of 2' -fucosyllactose using fucosyltransferase derived from Agrobacterium choreoides
<130> YW-19-001
<160> 7
<170> KoPatentIn 3.0
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<212> DNA
<213> Escherichia coli
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atgtcaaaag tcgctctcat caccggtgta accggacaag acggttctta cctggcagag 60
tttctgctgg aaaaaggtta cgaggtgcat ggtattaagc gtcgcgcatc gtcattcaac 120
accgagcgcg tggatcacat ttatcaggat ccgcacacct gcaacccgaa attccatctg 180
cattatggcg acctgagtga tacctctaac ctgacgcgca ttttgcgtga agtacagccg 240
gatgaagtgt acaacctggg cgcaatgagc cacgttgcgg tctcttttga gtcaccagaa 300
tataccgctg acgtcgacgc gatgggtacg ctgcgcctgc tggaggcgat ccgcttcctc 360
ggtctggaaa agaaaactcg tttctatcag gcttccacct ctgaactgta tggtctggtg 420
caggaaattc cgcagaaaga gaccacgccg ttctacccgc gatctccgta tgcggtcgcc 480
aaactgtacg cctactggat caccgttaac taccgtgaat cctacggcat gtacgcctgt 540
aacggaattc tcttcaacca tgaatccccg cgccgcggcg aaaccttcgt tacccgcaaa 600
atcacccgcg caatcgccaa catcgcccag gggctggagt cgtgcctgta cctcggcaat 660
atggattccc tgcgtgactg gggccacgcc aaagactacg taaaaatgca gtggatgatg 720
ctgcagcagg aacagccgga agatttcgtt atcgcgaccg gcgttcagta ctccgtgcgt 780
cagttcgtgg aaatggcggc agcacagctg ggcatcaaac tgcgctttga aggcacgggc 840
gttgaagaga agggcattgt ggtttccgtc accgggcatg acgcgccggg cgttaaaccg 900
ggtgatgtga ttatcgctgt tgacccgcgt tacttccgtc cggctgaagt tgaaacgctg 960
ctcggcgacc cgaccaaagc gcacgaaaaa ctgggctgga aaccggaaat caccctcaga 1020
gagatggtgt ctgaaatggt ggctaatgac ctcgaagcgg cgaaaaaaca ctctctgctg 1080
aaatctcacg gctacgacgt ggcgatcgcg ctggagtcat aa 1122
<210> 2
<211> 966
<212> DNA
<213> Escherichia coli
<400> 2
atgagtaaac aacgagtttt tattgctggt catcgcggga tggtcggttc cgccatcagg 60
cggcagctcg aacagcgcgg tgatgtggaa ctggtattac gcacccgcga cgagctgaac 120
ctgctggaca gccgcgccgt gcatgatttc tttgccagcg aacgtattga ccaggtctat 180
ctggcggcgg cgaaagtggg cggcattgtt gccaacaaca cctatccggc ggatttcatc 240
taccagaaca tgatgattga gagcaacatc attcacgccg cgcatcagaa cgacgtgaac 300
aaactgctgt ttctcggatc gtcctgcatc tacccgaaac tggcaaaaca gccgatggca 360
gaaagcgagt tgttgcaggg cacgctggag ccgactaacg agccttatgc tattgccaaa 420
atcgccggga tcaaactgtg cgaatcatac aaccgccagt acggacgcga ttaccgctca 480
gtcatgccga ccaacctgta cgggccacac gacaacttcc acccgagtaa ttcgcatgtg 540
atcccagcat tgctgcgtcg cttccacgag gcgacggcac agaatgcgcc ggacgtggtg 600
gtatggggca gcggtacacc gatgcgcgaa tttctgcacg tcgatgatat ggcggcggcg 660
agcattcatg tcatggagct ggcgcatgaa gtctggctgg agaacaccca gccgatgttg 720
tcgcacatta acgtcggcac gggcgttgac tgcactatcc gcgagctggc gcaaaccatc 780
gccaaagtgg tgggttacaa aggccgggtg gtttttgatg ccagcaaacc ggatggcacg 840
ccgcgcaaac tgctggatgt gacgcgcctg catcagcttg gctggtatca cgaaatctca 900
ctggaagcgg ggcttgccag cacttaccag tggttccttg agaatcaaga ccgctttcgg 960
gggtaa 966
<210> 3
<211> 1254
<212> DNA
<213> Escherichia coli
<400> 3
atgtactatt taaaaaacac aaacttttgg atgttcggtt tattcttttt cttttacttt 60
tttatcatgg gagcctactt cccgtttttc ccgatttggc tacatgacat caaccatatc 120
agcaaaagtg atacgggtat tatttttgcc gctatttctc tgttctcgct attattccaa 180
ccgctgtttg gtctgctttc tgacaaactc gggctgcgca aatacctgct gtggattatt 240
accggcatgt tagtgatgtt tgcgccgttc tttattttta tcttcgggcc actgttacaa 300
tacaacattt tagtaggatc gattgttggt ggtatttatc taggcttttg ttttaacgcc 360
ggtgcgccag cagtagaggc atttattgag aaagtcagcc gtcgcagtaa tttcgaattt 420
ggtcgcgcgc ggatgtttgg ctgtgttggc tgggcgctgt gtgcctcgat tgtcggcatc 480
atgttcacca tcaataatca gtttgttttc tggctgggct ctggctgtgc actcatcctc 540
gccgttttac tctttttcgc caaaacggat gcgccctctt ctgccacggt tgccaatgcg 600
gtaggtgcca accattcggc atttagcctt aagctggcac tggaactgtt cagacagcca 660
aaactgtggt ttttgtcact gtatgttatt ggcgtttcct gcacctacga tgtttttgac 720
caacagtttg ctaatttctt tacttcgttc tttgctaccg gtgaacaggg tacgcgggta 780
tttggctacg taacgacaat gggcgaatta cttaacgcct cgattatgtt ctttgcgcca 840
ctgatcatta atcgcatcgg tgggaaaaac gccctgctgc tggctggcac tattatgtct 900
gtacgtatta ttggctcatc gttcgccacc tcagcgctgg aagtggttat tctgaaaacg 960
ctgcatatgt ttgaagtacc gttcctgctg gtgggctgct ttaaatatat taccagccag 1020
tttgaagtgc gtttttcagc gacgatttat ctggtctgtt tctgcttctt taagcaactg 1080
gcgatgattt ttatgtctgt actggcgggc aatatgtatg aaagcatcgg tttccagggc 1140
gcttatctgg tgctgggtct ggtggcgctg ggcttcacct taatttccgt gttcacgctt 1200
agcggccccg gcccgctttc cctgctgcgt cgtcaggtga atgaagtcgc ttaa 1254
<210> 4
<211> 807
<212> DNA
<213> Unknown
<220>
<223> Pseudopedobacter saltans
<400> 4
atgatatttg taaccggata tggccagatg tgtaacaaca tccttcaatt tgggcatttc 60
tttgcttatg caaaaagaaa tggtttaaaa acggttggct tacgtttttg ctacaaatac 120
acttttttca agattagtaa cgaaaaaggc tataattggc cgacctatct ttatgcaaaa 180
tatggggcaa aaataggact tataaagtct gttgattttg acgaatcatt cgaaggtaca 240
aatgtagatt ctcttcaatt agacaaacaa accgtgttag ccaaaggctg gtattttaga 300
gactaccagg gatttcttaa ttaccgtaat gagcttaaag cacttttcga ctttaaagag 360
catattaaga aaccggtaga acagtttttt tcaacgttat caaaagacac catcaaagta 420
ggcctgcata taagacgtgg tgattataag acctggcacc agggtaaata cttttttagc 480
gacgaagaat acggtcaaat cgtaaattct tttgctaaaa gtttagataa accggtagaa 540
ttaattattg ttagcaatga tcccaaacta aacagcaaaa gttttgaaaa tttaacatcc 600
tgtaaagtat caatgttaaa tggcaatcct gccgaagatc tttaccttct ttctaaatgt 660
gattatatta ttggccctcc cagcactttt tctttaatgg cagcttttta cgaagaccgc 720
cctttatatt ggatatttga taaagaaaaa cagcttttag cagaaaactt tgacaagttc 780
gagaatctgt ttcgacacat tatttaa 807
<210> 5
<211> 268
<212> PRT
<213> Unknown
<220>
<223> Pseudopedobacter saltans
<400> 5
Met Ile Phe Val Thr Gly Tyr Gly Gln Met Cys Asn Asn Ile Leu Gln
1 5 10 15
Phe Gly His Phe Phe Ala Tyr Ala Lys Arg Asn Gly Leu Lys Thr Val
20 25 30
Gly Leu Arg Phe Cys Tyr Lys Tyr Thr Phe Phe Lys Ile Ser Asn Glu
35 40 45
Lys Gly Tyr Asn Trp Pro Thr Tyr Leu Tyr Ala Lys Tyr Gly Ala Lys
50 55 60
Ile Gly Leu Ile Lys Ser Val Asp Phe Asp Glu Ser Phe Glu Gly Thr
65 70 75 80
Asn Val Asp Ser Leu Gln Leu Asp Lys Gln Thr Val Leu Ala Lys Gly
85 90 95
Trp Tyr Phe Arg Asp Tyr Gln Gly Phe Leu Asn Tyr Arg Asn Glu Leu
100 105 110
Lys Ala Leu Phe Asp Phe Lys Glu His Ile Lys Lys Pro Val Glu Gln
115 120 125
Phe Phe Ser Thr Leu Ser Lys Asp Thr Ile Lys Val Gly Leu His Ile
130 135 140
Arg Arg Gly Asp Tyr Lys Thr Trp His Gln Gly Lys Tyr Phe Phe Ser
145 150 155 160
Asp Glu Glu Tyr Gly Gln Ile Val Asn Ser Phe Ala Lys Ser Leu Asp
165 170 175
Lys Pro Val Glu Leu Ile Ile Val Ser Asn Asp Pro Lys Leu Asn Ser
180 185 190
Lys Ser Phe Glu Asn Leu Thr Ser Cys Lys Val Ser Met Leu Asn Gly
195 200 205
Asn Pro Ala Glu Asp Leu Tyr Leu Leu Ser Lys Cys Asp Tyr Ile Ile
210 215 220
Gly Pro Pro Ser Thr Phe Ser Leu Met Ala Ala Phe Tyr Glu Asp Arg
225 230 235 240
Pro Leu Tyr Trp Ile Phe Asp Lys Glu Lys Gln Leu Leu Ala Glu Asn
245 250 255
Phe Asp Lys Phe Glu Asn Leu Phe Arg His Ile Ile
260 265
<210> 6
<211> 903
<212> DNA
<213> Helicobacter pylori
<400> 6
atggctttca aggtggtcca aatttgtggt ggacttggca atcagatgtt tcagtacgca 60
tttgcgaaaa gcctgcaaaa acattcgaat acgcctgtac tcctcgacat cacctcattt 120
gattggagcg atcggaaaat gcagctcgag cttttcccca ttgatctccc ctatgcgtct 180
gccaaggaga ttgccattgc gaaaatgcaa caccttccga aattggtacg tgacgcgctg 240
aaatgcatgg gattcgaccg tgtgtctcag gaaatcgtct ttgagtacga acctaagctg 300
ctgaagccat cgcgtttgac gtattttttc ggatactttc aagacccacg gtattttgac 360
gcaattagcc cacttattaa gcagacgttt actttgccac ccccaccaga aaacaacaag 420
aataacaaca agaaggagga agagtatcaa tgcaaactta gcttgatttt ggcagctaaa 480
aattcggtat ttgttcatat ccgccggggt gactatgtcg gaatcggatg ccaacttgga 540
atcgactacc agaaaaaggc tcttgagtac atggctaagc gtgtccccaa tatggaactc 600
ttcgtctttt gcgaggatct ggagttcacc cagaacctcg atctcggcta tccgttcatg 660
gacatgacca cccgcgacaa agaggaagaa gcatactggg atatgctgtt gatgcagtct 720
tgtcagcacg cgatcattgc aaattccact tactcatggt gggcggccta ccttattgaa 780
aatcctgaga aaattatcat cggaccaaag cactggctct ttggacacga aaacattctc 840
tgtaaggagt gggtgaagat cgaaagccat ttcgaagtga agagccagaa atacaatgct 900
taa 903
<210> 7
<211> 300
<212> PRT
<213> Helicobacter pylori
<400> 7
Met Ala Phe Lys Val Val Gln Ile Cys Gly Gly Leu Gly Asn Gln Met
1 5 10 15
Phe Gln Tyr Ala Phe Ala Lys Ser Leu Gln Lys His Ser Asn Thr Pro
20 25 30
Val Leu Leu Asp Ile Thr Ser Phe Asp Trp Ser Asp Arg Lys Met Gln
35 40 45
Leu Glu Leu Phe Pro Ile Asp Leu Pro Tyr Ala Ser Ala Lys Glu Ile
50 55 60
Ala Ile Ala Lys Met Gln His Leu Pro Lys Leu Val Arg Asp Ala Leu
65 70 75 80
Lys Cys Met Gly Phe Asp Arg Val Ser Gln Glu Ile Val Phe Glu Tyr
85 90 95
Glu Pro Lys Leu Leu Lys Pro Ser Arg Leu Thr Tyr Phe Phe Gly Tyr
100 105 110
Phe Gln Asp Pro Arg Tyr Phe Asp Ala Ile Ser Pro Leu Ile Lys Gln
115 120 125
Thr Phe Thr Leu Pro Pro Pro Pro Glu Asn Asn Lys Asn Asn Asn Lys
130 135 140
Lys Glu Glu Glu Tyr Gln Cys Lys Leu Ser Leu Ile Leu Ala Ala Lys
145 150 155 160
Asn Ser Val Phe Val His Ile Arg Arg Gly Asp Tyr Val Gly Ile Gly
165 170 175
Cys Gln Leu Gly Ile Asp Tyr Gln Lys Lys Ala Leu Glu Tyr Met Ala
180 185 190
Lys Arg Val Pro Asn Met Glu Leu Phe Val Phe Cys Glu Asp Leu Glu
195 200 205
Phe Thr Gln Asn Leu Asp Leu Gly Tyr Pro Phe Met Asp Met Thr Thr
210 215 220
Arg Asp Lys Glu Glu Glu Ala Tyr Trp Asp Met Leu Leu Met Gln Ser
225 230 235 240
Cys Gln His Ala Ile Ile Ala Asn Ser Thr Tyr Ser Trp Trp Ala Ala
245 250 255
Tyr Leu Ile Glu Asn Pro Glu Lys Ile Ile Ile Gly Pro Lys His Trp
260 265 270
Leu Phe Gly His Glu Asn Ile Leu Cys Lys Glu Trp Val Lys Ile Glu
275 280 285
Ser His Phe Glu Val Lys Ser Gln Lys Tyr Asn Ala
290 295 300