Production method of 3' -fucosyllactose using corynebacterium glutamicum
阅读说明：本技术 利用谷氨酸棒状杆菌的3’-岩藻糖基乳糖的生产方法 (Production method of 3' -fucosyllactose using corynebacterium glutamicum ) 是由 徐镇浩 郑相珉 李道行 全炯度 于 2018-04-20 设计创作，主要内容包括：本发明涉及一种利用野生型谷氨酸棒状杆菌菌株制造3’-岩藻糖基乳糖的方法,使用GRAS菌株即谷氨酸棒状杆菌(Corynebacterium glutamicum)菌株,可以安全地生产3’-岩藻糖基乳糖,并且可以高浓度、高收率、高生产率生产3’-岩藻糖基乳糖。(The present invention relates to a method for producing 3 '-fucosyllactose using a wild-type Corynebacterium glutamicum strain, which can produce 3' -fucosyllactose safely and with high concentration, high yield and high productivity using a GRAS strain, i.e., a Corynebacterium glutamicum (Corynebacterium glutamicum) strain.)
1. A recombinant Corynebacterium glutamicum (Corynebacterium glutamicum) is characterized in that,
converting to express alpha-1, 3-fucosyltransferase (alpha-1, 3-fucosyltransferase),
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),
phosphomannomutase (Phosphomannomutase) and GTP-mannose-1-phosphate guanylyltransferase (GTP-mannose-1-phosphate guanyltransferase) were retained.
2. The recombinant Corynebacterium glutamicum of claim 1, wherein the strain of the Corynebacterium glutamicum,
the above-mentioned alpha-1, 3-fucosyltransferase (alpha-1, 3-fucosyltransferase) is encrypted by the azolT gene.
3. The recombinant Corynebacterium glutamicum of claim 2, wherein the strain of the Corynebacterium glutamicum,
the aforementioned azolT gene is composed of the nucleic acid sequence of SEQ ID No. 5.
4. The recombinant Corynebacterium glutamicum of claim 1, wherein the strain of the Corynebacterium glutamicum,
the recombinant Corynebacterium glutamicum
Converted to overexpress phosphomannose mutase (Phosphomannomutase), and
the conversion is carried out to over-express GTP-mannose-1-phosphate guanine transferase (GTP-mannose-1-phosphate guanylyltransferase).
5. A production method of 3' -fucosyllactose is characterized in that,
culturing the recombinant Corynebacterium glutamicum (Corynebacterium glutamicum) according to claim 1 in a medium supplemented with lactose.
6. The method for producing 3' -fucosyllactose according to claim 5,
the medium further comprises glucose.
7. The method for producing 3' -fucosyllactose according to claim 6,
the above-mentioned production method of fucosyllactose is a batch culture or fed-batch culture in which glucose or lactose is additionally supplied.
Technical Field
The invention relates to a method for producing mutant microorganism and fucosyllactose from glucose and lactose by utilizing wild type corynebacterium glutamicum strain, in detail, the present invention is described in detail, relates to a method for producing 3' -fucosyllactose using a recombinant corynebacterium glutamicum in which phosphomannomutase (ManB), GTP-mannose-1-phosphate guanyltransferase (GTP-mannose-1-phosphate guanyltransferase, ManC), GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase, Gmd), GDP-L-fucose synthase (GDP-L-fucose synthase, Wcag), alpha-1, 3-fucosyltransferase (alpha-1, 3-fucosyltransferase) and a mutated lac operon from which lacZ is removed are introduced into the corynebacterium.
Background
Human breast Milk contains more than 200 kinds of oligosaccharides (HMOs) having different structures from each other, and is present at a considerably higher concentration (5 to 15g/L) than other mammals. The HMO has essential functions for infants such as prebiotic (prebiotic) effect, pathogenic bacteria intestinal adhesion inhibition effect, and immunoregulatory system regulation effect.
On the other hand, among HMOs, 3' -fucosyllactose is reported to be the main HMO involved in various biological activities. As a method for producing 3' -fucosyllactose, there are a method of directly extracting from breast milk and a method of synthesizing by a chemical or enzymatic method. However, the direct extraction method has disadvantages 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 use of toxic organic solvents. Further, the enzymatic synthesis method has disadvantages that GDP-L-fucose used as a fucose donor (donor) is very expensive and that fucosyltransferase (fucosyltransferase) is expensive to purify.
Therefore, direct extraction, chemical or enzymatic production methods are currently difficult to apply to the mass production of fucosyllactose. However, since fucosyllactose can be produced in large quantities from an inexpensive substrate by a simple process using a microbial bioengineering production method, attention is being given to a method for producing 3' -fucosyllactose which has a possibility of being developed into a health functional food and a raw material for pharmaceuticals.
On the other hand, the prior art for producing 3' -fucosyllactose by microorganisms is mostly a production technique using recombinant Escherichia coli. Coli used for experiments is not a pathogenic bacterium in fact, but it is widely recognized by consumers as a harmful bacterium and a cell membrane component may act as endotoxin, so that there is a disadvantage that separation and purification costs are high, and it is difficult to use e.coli as a host cell for 3' -fucosyllactose which is a raw material for producing foods and pharmaceuticals.
Korean patent No. 10-1544184 (granted date: 2015.08.21) relates to a mutant microorganism for producing 2 ' -fucosyllactose and a method for producing 2 ' -fucosyllactose using the same, and discloses a mutant microorganism in which one or more genes selected from the group consisting of an operon in which lacZ deformation or removal is introduced and a gene encoding FucT2 or a variant thereof, a gene encoding G6PDH (glucose-6-phosphate dehydrogenase) and GSK (guanosine-kinase) are introduced or amplified, and a method for producing 2 ' -fucosyllactose using the same.
Korean patent No. 10-1648352 (grant date: 2016.08.09) relates to a method in which at least one of genes encoding fucose isomerase (fuco isomerase, FucI), fucokinase (FucK) and fuco1-phosphate aldolase (FucA), which are fucose metabolizing enzymes, is disrupted, and a method for producing fucosyllactose using recombinant Escherichia coli for producing fucosyllactose, which retains "a lacZ gene encoding a beta-galactosidase having a lower activity than wild-type beta-galactosidase, a lac operator composed of a wild-type lacY gene and a wild-type lacA gene" or "a lac operator completely removed from a wild-type lacZ gene and composed of only a wild-type lacY gene and a wild-type lacA gene" instead of the "wild-type lac operator", and which can produce 2-or 3-fucosyllactose with high productivity.
Disclosure of Invention
Technical subject
The purpose of the present invention is to develop and provide a method for producing 3' -fucosyllactose at high concentration, high yield and high productivity using Corynebacterium glutamicum (Corynebacterium glutamicum) which is safer than escherichia coli, as a host cell for fucosyllactose, which is a raw material for producing foods and pharmaceuticals.
Means for solving the problems
The invention provides a recombinant Corynebacterium glutamicum (Corynebacterium glutamicum), which is characterized in that the recombinant Corynebacterium glutamicum is converted to express alpha-1, 3-fucosyltransferase (alpha-1, 3-fucosyltransferase), converted to express GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase), converted to express GDP-L-fucose synthase (GDP-L-fucose synthase), converted to express lactose permease (lactose permease), and reserved phosphomannose mutase (phosphomannose) and GTP-mannose-1-phosphoguanine transferase (GTP-mannose-1-phosphoguanine transferase).
In the recombinant Corynebacterium glutamicum (Corynebacterium glutamicum) of the present invention, the above-mentioned alpha-1, 3-fucosyltransferase (alpha-1, 3-fucosyltransferase) is preferably used as encrypted with an azo T gene. In this case, the aforementioned azolt gene may preferably be composed of the nucleic acid sequence of sequence No. 5.
In the recombinant Corynebacterium glutamicum (Corynebacterium glutamicum) of the present invention, preferably, the recombinant Corynebacterium glutamicum is transformed to overexpress Phosphomannomutase (Phosphomannomutase) and to overexpress GTP-mannose-1-phosphoguanyltransferase (GTP-mannose-1-phosphoguanyltransferase).
The invention provides a production method of 3' -fucosyllactose, which is characterized in that recombinant Corynebacterium glutamicum (Corynebacterium glutamicum) is cultured in a culture medium added with lactose, the recombinant Corynebacterium glutamicum (Corynebacterium glutamicum) is converted to express alpha-1, 3-fucosyltransferase (alpha-1, 3-fucosyltransferase), converted to express GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase), converted to express GDP-L-fucose synthase (GDP-L-fucose synthase), converted to express lactose permease (lactonase), and retained phosphomannose mutase (phosphomannose) and GTP-mannose-1-phosphate guanine transferase (GTP-mannose-1-phosphoguanine transferase).
In the 3' -fucosyllactose production method of the present invention, preferably, the culture medium further contains glucose. In this case, the fucosyllactose may be produced by a batch culture or a fed-batch culture in which glucose or lactose is additionally supplied.
Effects of the invention
According to the present invention, 3 '-fucosyllactose can be produced by using a GRAS strain, i.e., Corynebacterium glutamicum (Corynebacterium glutamicum) strain, and 3' -fucosyllactose can be produced safely compared to conventional Escherichia coli. Also, when the Corynebacterium glutamicum strain of the present invention is used, 3' -fucosyllactose can be produced at high concentration, high yield, and high productivity.
Drawings
FIG. 1 is a schematic representation of the metabolic pathways introduced for the biosynthesis of GDP-L-fucose and 3' -fucosyllactose from C.glutamicum strains.
FIG. 2 is a result of measuring 3' -fucosyllactose produced in Corynebacterium glutamicum pVBCL + pEGWA (pEGW + azoT) by HPLC.
FIG. 3 is a graph showing the results of batch culture in flasks using recombinant Corynebacterium glutamicum (C.glutamicum) pVBCL + pEGWA. When Optical Density (OD)600) To aboutAt 0.8, IPTG and lactose were added to give final concentrations of 1.0mM, 10g/L, respectively (arrows). The symbols in the figures are as follows: ●: dry cell weight, a: glucose, ■: lactose, xxx: lactate (lactate),. diamond solid: 3' -fucosyllactose.
FIG. 4 is a graph showing the results of fed-batch culture using recombinant Corynebacterium glutamicum (C.glutamicum) pVBCL + pEGWT. After the total consumption of 40g/L of glucose initially charged, the continuous feeding of glucose was started and IPTG and lactose were added (arrows). The symbols in the figures are as follows: ●: dry cell weight, a: glucose, ■: lactose, xxx: lactate,. diamond: 3' -fucosyllactose.
Detailed Description
The invention provides a recombinant Corynebacterium glutamicum (Corynebacterium glutamicum), which is characterized in that the recombinant Corynebacterium glutamicum is converted to express alpha-1, 3-fucosyltransferase (alpha-1, 3-fucosyltransferase), converted to express GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase), converted to express GDP-L-fucose synthase (GDP-L-fucose synthase), converted to express lactose permease (lactose permease), and reserved phosphomannose mutase (phosphomannose) and GTP-mannose-1-phosphoguanine transferase (GTP-mannose-1-phosphoguanine transferase).
The present inventors previously applied for a method for producing 3' -fucosyllactose using E.coli by Korean patent application No. 10-2016-0012803 (2016.02.02). However, it has been pointed out many times that when 3' -fucosyllactose is used as a functional food additive, there are some problems in the production thereof by E.coli due to various safety problems possessed by E.coli. Therefore, the present invention attempts to produce 3' -fucosyllactose by a substitute strain that has no problem in food safety.
In the present invention, Corynebacterium glutamicum (Corynebacterium glutamicum) was selected as a host cell for producing 3' -fucosyllactose, and unlike conventionally used escherichia coli, this strain is not only a strain identified as gras (genetically recovered as safe), but also a strain that does not produce endotoxin and is widely used for industrial production of amino acids and nucleic acids as food additives. Therefore, Corynebacterium glutamicum is a strain suitable for producing food and pharmaceutical products, and has an advantage that it can eliminate the safety concern of consumers.
However, the strains of Escherichia coli and Corynebacterium glutamicum themselves differ in their genetic characteristics, and it is therefore necessary to use strategies different from those applied to Escherichia coli. In order to produce 3' -fucosyllactose, both E.coli and C.glutamicum basically require the introduction of an exogenous alpha-1, 3-fucosyltransferase (alpha-1, 3-fucosyltransferase) enzyme, however, Corynebacterium glutamicum needs to further introduce GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase, Gmd), GDP-L-fucose synthase (GDP-L-fucose synthase, which is also called "GDP-4-keto-6-deoxy-D-mannose-3, 5-epimerase-4-reductase", and, abbreviated as "Wcag", and a gene encrypting the enzyme is particularly called "Wcag"), lactose permease (lactose permease, LacY). That is, Escherichia coli has genes encoding GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase, Gmd), GDP-L-fucose synthase (GDP-L-fucose synthase, Wcag), and lactose permease (LacY), but Corynebacterium glutamicum does not have a gene encoding the above enzymes, and therefore, it is necessary to introduce and express them from the outside.
At this time, preferably, the gene (azoT) derived from Azospirillum brasilense (Azospirillum brasilense) is preferably used as the gene for encrypting the above-mentioned alpha-1, 3-fucosyltransferase (alpha-1, 3-fucosyltransferase). Further, as the gene encoding GDP-D-mannose-4,6-dehydratase (GDP-D-mannose-4,6-dehydratase, Gmd), GDP-L-fucose synthase (GDP-L-fucose-synthase, Wcag) and lactose permease (LacY), a gene derived from Escherichia coli was used.
On the other hand, it is preferable that the recombinant Corynebacterium glutamicum of the present invention is transformed to overexpress Phosphomannomutase (Phosphomannomutase) and to overexpress GTP-mannose-1-phosphate guanylyltransferase (GTP-mannose-1-phosphate guanylyltransferase). Corynebacterium glutamicum itself may retain and express genes encoding Phosphomannomutase (ManB), GTP-mannose-1-phosphate guanylyltransferase (GTP-mannose-1-phosphate guanylyltransferase, ManC), and thus it is not necessary to introduce genes encoding such enzymes, but it is necessary to overexpress such genes for mass production. Therefore, in the present invention, it is desirable to convert Corynebacterium glutamicum to a form capable of overexpressing both of these enzymes.
On the other hand, the action of the enzyme can be understood from FIG. 1, and thus, the detailed description thereof is omitted. However, it is specifically stated that lactose permease (LacY) is an enzyme involved in the transport of lactose present outside the strain to the inside of the strain. In the following examples of the present invention, experiments were conducted after introducing lacYA gene in which lacZ was removed from Lac operon of E.coli, but in the present invention, Lac operon was introduced for the purpose of introducing lactose, and thus lacY gene alone, not lacA gene, was introduced.
On the other hand, the term "expression" used in the present invention means that an enzyme which cannot be expressed by the C.glutamicum strain of the present invention itself is expressed, and a foreign gene is introduced into the strain to express the enzyme artificially, and the term "overexpression" means that the C.glutamicum strain of the present invention itself has a gene encoding the corresponding enzyme and can express the enzyme by itself, but increases the expression level thereof for mass production, and artificially increases the expression level of the corresponding enzyme to overexpress the enzyme.
On the other hand, the present inventors confirmed that 3' -fucosyllactose, which is a breast milk oligosaccharide, can be produced in large quantities from corynebacterium glutamicum (c.glutamicum) by the above-described transformation strategy.
On the other hand, in the present invention, a gene encoding the α -1,3-fucosyltransferase (α -1,3-fucosyltransferase) is preferably a gene encrypted with an azolt gene, and the azolt gene may preferably be composed of the nucleic acid sequence of seq id No. 5. In order to produce α -1, 3-fucosyllactose, α -1,3-fucosyltransferase (α -1,3-fucosyltransferase) which performs α -1, 3-fucosyllactose production reaction with GDP-L-fucose and (GDP-L-fucose) and lactose (lactose) substrates is required (see fig. 1). This enzyme is present in various microorganisms, and in the present invention, a gene (azoT) derived from Azospirillum brasilense is used. When α -1,3-fucosyltransferase from other sources was used, the yield of 3 '-fucosyllactose was negligible, but when the azoT gene was used, the yield of 3' -fucosyllactose was significantly increased compared to when other sources were used.
In another aspect, the present invention provides a method for producing 3' -fucosyllactose, comprising culturing the recombinant Corynebacterium glutamicum of the present invention in a medium supplemented with lactose. When the recombinant Corynebacterium glutamicum strain of the present invention is used, 3' -fucosyllactose can be produced at high concentration, high yield and high productivity.
On the other hand, in the above-described method for producing 3' -fucosyllactose of the present invention, the culture medium preferably further contains glucose. By adding glucose to the medium, the growth of the strain becomes active, and 3' -fucosyllactose can be produced with higher productivity.
On the other hand, the above-mentioned method for producing 3' -fucosyllactose according to the present invention can be carried out by fed-batch culture in which lactose is supplied in batch or in addition. As for the specific details of the batch or fed-batch culture, those known in the art can be used, and the description thereof is omitted.
On the other hand, the strain Corynebacterium glutamicum of the present invention introduced lactose permease (lactose permease) for the purpose of introducing lactose, which is a substrate for 3' -FL production, into the cells. That is, in order to produce 3' -FL using Corynebacterium glutamicum, it is necessary to transform it with a lactose-penetrating enzyme capable of introducing lactose into the cells of the strain of the present invention transformed with the enzyme.
However, lactose permeases are usually "glucose repressed" in the presence of glucose, thereby inhibiting their activity. As a result, the introduction of lactose did not occur in the presence of glucose, and 3-FL could not be produced finally.
However, the "glucose repression" action does not occur in C.glutamicum used as a host strain for producing 3' -FL in the present invention, and therefore 3' -FL can be produced according to the introduction of lactose even in the presence of glucose, whereby the productivity of 3' -FL can be maximized.
On the other hand, it was confirmed by the following experiments of the present invention that 3 '-FL is produced in the "nonfocal-associated product formation" production mode by the method for producing 3' -FL of Corynebacterium glutamicum of the present invention.
The "nonassociates product formation" produced independently of the growth of the host strain does not require the growth of the host strain for producing the metabolite at the same time, and therefore has an advantage that the metabolite can be produced in large quantities in a short time by throwing the substrate after culturing the host strain in large quantities. Also, the host strain used in the culture can be reused, thus having an advantage of maximizing productivity. Therefore, in the present invention, the 3 '-FL production method constructed using Corynebacterium glutamicum is a method capable of maximizing the production of 3' -FL.
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.
Example 1: preparation of recombinant Strain and plasmid
Cloning Escherichia coli (Escherichia coli TOP10) was used for plasmid preparation, and Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC 13032 was used for production of 3' -fucosyllactose (3 ' -fucosyllactise, 3' -FL). Plasmids for pVBCL expression were used which expressed the manB and manC and lacYA gene clusters developed from previous studies. Furthermore, a vector was constructed for expressing α -1,3-fucosyltransferase (α -1,3-fucosyltransferase) (azot)) in a plasmid for expression of pEGW expressing the gmd and wcAG genes. At this time, α -1,3-fucosyltransferase (azoT) was derived from Azospirillum brasilense ATCC 29145, and α -1,3-fucosyltransferase (azoT) was inserted into pEGW vector using the restriction enzyme of
On the other hand, the gene sequences, strains, plasmids, and oligonucleotides of manB, manC, gmd, WcaG, lacYA, and α -1,3-fucosyltransferase (azoT) used above are shown in tables 1 to 3 below.
[ Table 1]
Name of gene
Serial number
manB
Sequence No. 1
manC
Sequence number 2
gmd-wcaG
Sequence No. 3
lacYA
Sequence number 4
Sequence number 5
[ Table 2]
[ Table 3]
Example 2: production of 3' -fucosyllactose Using recombinant Corynebacterium glutamicum
(1) Culture conditions and methods
A test tube containing 5mL of BHI (brain Heart infusion) medium containing an appropriate antibiotic (kanamycin 25. mu.g/mL,
In a container containing 100mL ((NH)4)2SO4 20g/L，urea 5g/L，KH2PO4 1g/L，K2HPO4 1g/L，MgSO40.25g/L，MOPS 42g/L，CaCl2 10mg/L，Biotin 0.2mg/L，Protocatechuic acid 30mg/L，FeSO47H20 10mg/L，MnSO4H2O 10mg/L，ZnSO47H2O 1mg/L，CuSO4 0.2mg/L，NiCl26H2O0.02 mg/L, pH7.0) was placed in a 250ml flask and batch-cultured at 30 ℃. The stirring speed was maintained at 250rpm and the culture was performed. In batch culture, at Optical Density (OD)600) When the concentration reached 0.8, IPTG (isopropyl-. beta. -D-thiogalactopyranoside) and lactose were added to the mixture so that the final concentrations reached 1.0mM and 10g/L, respectively.
Fed-batch culture for high concentration cell culture was performed in a 2.5L volume bioreactor (bioreactor, Kobiotech, Incheon, Korea) containing 1.0L of minimal medium containing 40g/L glucose and appropriate antibiotics (kanamycin 25. mu.g/mL,
After the initial addition of glucose was completely consumed, a feed solution (feeding solution) containing 800g/L of glucose was supplied at a rate of 5.7g/L/h by a continuous feeding method. Meanwhile, in order to direct tac promoter mediated gene expression to produce 3' -fucosyllactose, IPTG, lactose were added so that the final concentration reached 1.0mM, 10 g/L.
During the fermentation, 28% NH is automatically supplied if the pH of the medium is below the set-point4OH, if above set point, 2N HCl is added to maintain the pH within a certain range (pH 6.98-7.02). The pH of the medium was measured in real time using a pH electrode (Mettler Toledo, USA). To prevent oxygen deficiency, the stirring speed and aeration speed were maintained at 1000rpm and 2vvm, respectively.
(2) Determination of the concentration of cells and metabolites
The Optical Density (OD) multiplied by the previously measured transformation constant 0.3 determines the dry cell weight. After the sample was appropriately diluted so that the optical density was adjusted to a range between 0.1 and 0.5, the Optical Density (OD) was measured at an absorbance of 600nm using a spectrophotometer (spectrophotometer, Ultrospec 2000, Amersham Pharmacia Biotech, USA).
The concentrations of 3' -fucosyllactose, lactose, lactate, glucose and acetic acid were measured using an hplc (high performance chromatography) equipped with a "Carbohydrate Analysis column (Rezex ROA-organic acid, Phenomenex, USA)" and "ri (reactive index)" detector. 20 μ l of the medium diluted 10-fold was analyzed using a heated chromatography column at 60 ℃. At a flow rate of 0.6mL/min, 5mM H was used as the mobile phase2SO4And (3) solution.
(3) Production of 3' fucosyllactose by batch culture
To confirm the 3' -fucosyllactose production performance and fermentation characteristics, recombinant corynebacterium glutamicum introduced with ManB, ManC, Gmd, WcaG, azoT, and lac operon (lacYA) from which lacZ was removed was batch-cultured in flasks, respectively. At Optical Density (OD)600) When the concentration reached 0.8, IPTG and lactose were added so that the final concentrations reached 1.0mM and 10g/L, respectively.
As a result of the batch culture in the flask, 390mg/L of 3' -fucosyllactose was produced, and the yield of 2 ' -fucosyllactose to lactose was 0.32mole of 2 ' -fucosyllactose/mole of lactose, and the productivity was 5.49mg/L/h (FIG. 3 and Table 4). FIG. 2 is a result of measuring 3' -fucosyllactose produced in Corynebacterium glutamicum pVBCL + pEGWA (pEGW + azoT) by HPLC. The results of the above batch culture are described in table 4 below, and fig. 3 is a graph showing the results of the batch culture in flasks using recombinant corynebacterium glutamicum (c.glutamicum) pvgcl + pegfa.
[ Table 4]
Results of batch culture in flasks Using recombinant Corynebacterium glutamicum (C.glutamicum) pVBCL + pEGWA
(4) Production of 3' fucosyllactose by fed batch culture
In order to produce high-concentration 3' -fucosyllactose by high-concentration cell culture, fed-batch culture was performed in a 2.5L-level fermenter using recombinant corynebacterium glutamicum (c.glutamicum) into which pvgcl, a pegfa plasmid was introduced.
From the time when the initially added 40g/L glucose was completely consumed, the feed solution (feeding solution) was supplied at a rate of 5.7g/L/h by a continuous feeding method to maintain the cell growth. At the same time, IPTG and lactose were added in order to guide the production of 3' -fucosyllactose.
As a result of the experiment, acetic acid was not produced at all during the fermentation, and the cells finally reached a dry cell weight of 48.9g/L by the metabolism of glucose. The maximum 3 '-fucosyllactose concentration was 3.6g/L, the production yield to lactose was 0.17mole of 3' -fucosyllactose/mole of lactose, and the productivity was 0.03g/L/h (FIG. 4 and Table 5).
The results of fed-batch culture for producing 3' -fucosyllactose are described in the following Table 5, and FIG. 4 is a graph showing the results of fed-batch culture using recombinant Corynebacterium glutamicum pVBCL + pEGWA.
[ Table 5]
Fed-batch culture results in flasks Using recombinant Corynebacterium glutamicum (C.glutamicum) pVBCL + pEGWA
<110> department of university of seoul university cooperative group
<120> recombinant Corynebacterium glutamicum for producing 3 '-fucosyllactose and method for producing 3' -fucosyllactose therefrom
<130> YP-18-055
<150> KR 10-2017-0051871
<151> 2017-04-21
<160> 7
<170> KoPatentIn 3.0
<210> 1
<211> 1377
<212> DNA
<213> Unknown
<220>
<223> Corynebacterium glutamicum ATCC 13032
<400> 1
atgcgtaccc gtgaatctgt cacggctgta attaaggcgt atgacgtccg tggtgttgtt 60
ggtgtcgata ttgatgctga tttcatttct gagactggcg ctgcctttgg tcggctcatg 120
cgtagtgagg gtgaaaccac cgttgctatt ggccatgaca tgcgtgattc ctcccctgaa 180
ttggccaagg cgtttgccga tggcgtgact gcacagggtt tggatgttgt tcatttggga 240
ctgacttcta ctgatgagct gtactttgcg tccggaacct tgaagtgtgc tggtgcgatg 300
tttactgcgt cgcataaccc cgctgagtac aacggcatca agttgtgtcg tgcgggtgct 360
cgtccggtcg gtcaggattc tggtttggcc aacatcattg atgatctggt tgagggtgtt 420
ccagcgtttg atggtgagtc aggttcggtt tctgagcagg atttgctgag cgcatatgcc 480
gagtacctca atgagcttgt tgatctgaag aacatccgcc cgttgaaggt tgctgtggat 540
gcggcaaacg gcatgggtgg gttcactgtc cctgaggtat tcaagggtct gccacttgat 600
gttgcgccac tgtattttga gcttgacggc aatttcccca accatgaggc caatcctctg 660
gagcctgcca acctggttga tttgcagaag tttaccgtag agaccggatc tgatatcggt 720
ttggcgttcg acggcgatgc ggatcgttgc ttcgtggtcg atgagaaggg ccagccagtc 780
agcccttcgg cgatctgtgc gatcgtagcg gagcgttact tggagaagct tccgggttcc 840
accatcatcc acaacctgat tacctctaag gctgtgcctg aggtgattgc tgaaaacggt 900
ggcactgcgg tgcgtactcg cgtgggtcac tccttcatca aggcgaagat ggcagagacc 960
ggtgcggcct ttggtggcga gcactctgcg cactactact tcactgagtt cttcaatgcg 1020
gactccggca ttttggctgc gatgcacgtg ctggctgcgc tgggaagcca ggaccagcca 1080
ctcagtgaga tgatggctag gtataaccgg tacgttgctt caggcgagtt gaactcccgt 1140
ttggctaatg cagaggcgca gcaagagcgc acccaggctg tgctcgatgc gttcgctgat 1200
cgcaccgagt ccgtggacac ccttgacggc gtgactgtgg aactcaagga cacctccgcg 1260
tggttcaacg tgcgtgcgtc caacaccgag ccgctgcttc gcctcaatgt tgaagctgca 1320
tcgaaggaag aagtcgatgc gttggtagcg gagattctag ggattatccg cgcataa 1377
<210> 2
<211> 1089
<212> DNA
<213> Unknown
<220>
<223> Corynebacterium glutamicum ATCC 13032
<400> 2
atgactttaa ctgacaacag caaaaacgtt gatgctgtca tcttggtcgg tggcaaaggt 60
acccgactgc gccccctgac cgtcaatact ccaaagccaa tgctgccaac tgctggccac 120
ccattcttga cccacctttt ggcccgcatc aaggccgcag gcatcacaca cgtcgtgctg 180
ggaacgtcat tcaaagctga agtcttcgag gaatacttcg gagatggctc cgaaatgggc 240
ttggaaattg aatatgtcgt cgaggatcag cctttgggca ctggtggtgg catccgaaac 300
gtctacgaca agctgcgtca cgatactgcg attgtgttca acggcgatgt gctctccggt 360
gcggatctca acagcattct ggacacccac cgcgaaaagg acgcagatct gaccatgcat 420
ctcgtgcgcg tagctaaccc tcgtgcgttt ggttgcgtcc ccaccgatga ggatggtcgc 480
gtcagcgaat tccttgaaaa gaccgaagat ccaccaaccg atcagatcaa cgccggctgc 540
tacgtgttca agaaggaact catcgagcag atcccggcag gccgagcagt ttccgtcgag 600
cgcgaaacct tccctcagct gttggaagaa ggcaagcgag tcttcggcca cgtcgacgct 660
tcctactggc gcgacatggg caccccaagc gacttcgtcc gcggctcggc tgacctggtc 720
cgcggcattg cgtactcccc attgctcgaa ggcaaaacag gagagtcgct tgtcgacgcc 780
tccgccggcg ttcgcgacgg cgtcctgctg ctcggcggaa ccgtagtcgg ccgcggcact 840
gagatcggtg ccggctgccg cgttgacaac actgttattt tcgacggcgt caccattgaa 900
ccaggtgcgg tcattgaaaa ttccatcatt tcctcgggag cacgcatcgg tgctaatgcg 960
cacatctccg gttgcatcat tggcgagggc gcacaggttg gtgctcggtg tgaactcaac 1020
gcagggatgc gcgtcttccc aggcgttgtg atcccagaca gcggaattcg tttttcgtct 1080
gatcagtag 1089
<210> 3
<211> 2090
<212> DNA
<213> Unknown
<220>
<223> E.coli K-12 MG1655
<400> 3
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 aagcatgagt aaacaacgag 1140
tttttattgc tggtcatcgc gggatggtcg gttccgccat caggcggcag ctcgaacagc 1200
gcggtgatgt ggaactggta ttacgcaccc gcgacgagct gaacctgctg gacagccgcg 1260
ccgtgcatga tttctttgcc agcgaacgta ttgaccaggt ctatctggcg gcggcgaaag 1320
tgggcggcat tgttgccaac aacacctatc cggcggattt catctaccag aacatgatga 1380
ttgagagcaa catcattcac gccgcgcatc agaacgacgt gaacaaactg ctgtttctcg 1440
gatcgtcctg catctacccg aaactggcaa aacagccgat ggcagaaagc gagttgttgc 1500
agggcacgct ggagccgact aacgagcctt atgctattgc caaaatcgcc gggatcaaac 1560
tgtgcgaatc atacaaccgc cagtacggac gcgattaccg ctcagtcatg ccgaccaacc 1620
tgtacgggcc acacgacaac ttccacccga gtaattcgca tgtgatccca gcattgctgc 1680
gtcgcttcca cgaggcgacg gcacagaatg cgccggacgt ggtggtatgg ggcagcggta 1740
caccgatgcg cgaatttctg cacgtcgatg atatggcggc ggcgagcatt catgtcatgg 1800
agctggcgca tgaagtctgg ctggagaaca cccagccgat gttgtcgcac attaacgtcg 1860
gcacgggcgt tgactgcact atccgcgagc tggcgcaaac catcgccaaa gtggtgggtt 1920
acaaaggccg ggtggttttt gatgccagca aaccggatgg cacgccgcgc aaactgctgg 1980
atgtgacgcg cctgcatcag cttggctggt atcacgaaat ctcactggaa gcggggcttg 2040
ccagcactta ccagtggttc cttgagaatc aagaccgctt tcgggggtaa 2090
<210> 4
<211> 3335
<212> DNA
<213> Unknown
<220>
<223> E.coli BL21star(DE3)
<400> 4
accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa 60
ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc 120
tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg 180
gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa 240
ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg 300
ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg 360
gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc 420
gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct 480
gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc 540
atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc 600
gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt 660
ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa 720
cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag 780
ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc 840
gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat 900
accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg 960
ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc 1020
aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa 1080
accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga 1140
ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc 1200
ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 1260
atttcacaca ggaaacagct atgtactatt taaaaaacac aaacttttgg atgttcggtt 1320
tattcttttt cttttacttt tttatcatgg gagcctactt cccgtttttc ccgatttggc 1380
tacatgacat caaccatatc agcaaaagtg atacgggtat tatttttgcc gctatttctc 1440
tgttctcgct attattccaa ccgctgtttg gtctgctttc tgacaaactc gggctgcgca 1500
aatacctgct gtggattatt accggcatgt tagtgatgtt tgcgccgttc tttattttta 1560
tcttcgggcc actgttacaa tacaacattt tagtaggatc gattgttggt ggtatttatc 1620
taggcttttg ttttaacgcc ggtgcgccag cagtagaggc atttattgag aaagtcagcc 1680
gtcgcagtaa tttcgaattt ggtcgcgcgc ggatgtttgg ctgtgttggc tgggcgctgt 1740
gtgcctcgat tgtcggcatc atgttcacca tcaataatca gtttgttttc tggctgggct 1800
ctggctgtgc actcatcctc gccgttttac tctttttcgc caaaacggat gcgccctctt 1860
ctgccacggt tgccaatgcg gtaggtgcca accattcggc atttagcctt aagctggcac 1920
tggaactgtt cagacagcca aaactgtggt ttttgtcact gtatgttatt ggcgtttcct 1980
gcacctacga tgtttttgac caacagtttg ctaatttctt tacttcgttc tttgctaccg 2040
gtgaacaggg tacgcgggta tttggctacg taacgacaat gggcgaatta cttaacgcct 2100
cgattatgtt ctttgcgcca ctgatcatta atcgcatcgg tgggaaaaac gccctgctgc 2160
tggctggcac tattatgtct gtacgtatta ttggctcatc gttcgccacc tcagcgctgg 2220
aagtggttat tctgaaaacg ctgcatatgt ttgaagtacc gttcctgctg gtgggctgct 2280
ttaaatatat taccagccag tttgaagtgc gtttttcagc gacgatttat ctggtctgtt 2340
tctgcttctt taagcaactg gcgatgattt ttatgtctgt actggcgggc aatatgtatg 2400
aaagcatcgg tttccagggc gcttatctgg tgctgggtct ggtggcgctg ggcttcacct 2460
taatttccgt gttcacgctt agcggccccg gcccgctttc cctgctgcgt cgtcaggtga 2520
atgaagtcgc ttaagcaatc aatgtcggat gcggcgcgag cgccttatcc gaccaacata 2580
tcataacgga gtgatcgcat tgaacatgcc aatgaccgaa agaataagag caggcaagct 2640
atttaccgat atgtgcgaag gcttaccgga aaaaagactt cgtgggaaaa cgttaatgta 2700
tgagtttaat cactcgcatc catcagaagt tgaaaaaaga gaaagcctga ttaaagaaat 2760
gtttgccacg gtaggggaaa acgcctgggt agaaccgcct gtctatttct cttacggttc 2820
caacatccat ataggccgca atttttatgc aaatttcaat ttaaccattg tcgatgacta 2880
cacggtaaca atcggtgata acgtactgat tgcacccaac gttactcttt ccgttacggg 2940
acaccctgta caccatgaat tgagaaaaaa cggcgagatg tactcttttc cgataacgat 3000
tggcaataac gtctggatcg gaagtcatgt ggttattaat ccaggcgtca ccatcgggga 3060
taattctgtt attggcgcgg gtagtatcgt cacaaaagac attccaccaa acgtcgtggc 3120
ggctggcgtt ccttgtcggg ttattcgcga aataaacgac cgggataagc actattattt 3180
caaagattat aaagttgaat cgtcagttta aattataaaa attgcctgat acgctgcgct 3240
tatcaggcct acaagttcag cgatctacat tagccgcatc cggcatgaac aaagcgcagg 3300
aacaagcgtc gcatcatgcc tctttgaccc acagc 3335
<210> 5
<211> 978
<212> DNA
<213> Unknown
<220>
<223> Azospirillum brasilense ATCC 29145
<400> 5
atgctcgatc agcggacaag cgcgtttctt gaggaattcc tggcgaagcc gggcggcgat 60
cccgagcggc tcgaccgctt cctgctgcac ggcccgtacc gcggccggcg cggcggcaaa 120
ccgcggctga agctggcctt ccacgacttc tggccggagt tcgacaaggg cacgaacttc 180
ttcatcgaga tcctgtccag ccgcttcgac ctgtcggtgg tcgaggacga cagcgacctc 240
gccatcgtgt cggtcttcgg cgggcggcac cgcgaggcgc gcagccgccg caccctgttc 300
ttcaccgggg agaacgtgcg cccgccgttg gacggcttcg acatggcggt gtccttcgac 360
cgcgtcgacg acccgcgcca ttaccgcctg ccgctctacg tcatgcacgc ctacgagcac 420
atgcgggagg gggcggtgcc gcatttctgt tcgccggtcc tgccgccggt gccgccgacg 480
cgggcggcct tcgcggagcg cggcttctgc gccttcctct acaagaaccc gaacggggag 540
cgccgcaacc gcttcttccc ggtgctggac gggcggcggc gcgtcgattc ggtgggctgg 600
cacctgaaca acaccggcag cgtcgtcaag atgggctggc tgtcgaagat ccgcgtcttc 660
gaacgctacc gtttcgcctt cgccttcgag aacgccagcc atcccggcta tctgacggaa 720
aagatcctgg acgtcttcca ggccggggcg gtgccgctct attggggtga tcccgacctg 780
gagcgcgagg tggcggtcgg cagcttcatc gacgtgtcgc gcttcgccac ggacgaggag 840
gcggtggacc acatccttgc ggtggacgac gattacgacg cctattgcgc ccaccgcgcc 900
gtggcgccct tcctggggac ggaggagttt tatttcgacg cctaccgcct cgccgactgg 960
atcgagagcc ggctgtaa 978
<210> 6
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> F_inf_sac1_azoT
<400> 6
gctttcgggg gtaagagctc aaggagatat acaatgctcg atcagcggac aagc 54
<210> 7
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223> R_inf_sac1_azoT
<400> 7
cggccagtga attcgagctc ttacagccgg ctctcgatcc 40
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