CN115838682A - Bacillus licheniformis engineering strain for efficiently producing 2' -fucosyllactose by utilizing mannan - Google Patents
Bacillus licheniformis engineering strain for efficiently producing 2' -fucosyllactose by utilizing mannan Download PDFInfo
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Abstract
The invention discloses a bacillus licheniformis engineering strain for efficiently producing 2' -fucosyllactose by utilizing mannan, belonging to the technical field of synthetic biology and microbial metabolic engineering. The invention uses food safe microbe Bacillus licheniformis as host cell, uses the promoter with quorum sensing effect to express mannose-specific phosphotransferase transporter (E.coli), phosphomannose mutase (E.coli), mannose-1-phosphate-guanylyltransferase (E.coli), GDP-mannose-4,6-dehydratase (E.coli), GDP-L-fucose synthetase (E.coli) and fucosyltransferase (helicobacter pylori). The recombinant bacterium has the ability to synthesize 2' -FL by using inexpensive mannan such as konjac flour or corn flour. Compared with the synthesis route based on fine carbon sources such as glucose, glycerol and the like, the new synthesis route has obvious advantages in production efficiency and cost.
Description
Technical Field
The invention provides a bacillus licheniformis engineering strain for efficiently producing 2' -fucosyllactose by utilizing mannan, belonging to the technical field of synthetic biology and microbial metabolic engineering.
Background
The composition of intestinal microorganisms can affect human health. Breast milk is the most important food for infants and contains specific ingredients, such as oligosaccharides, antibodies and vitamins, that affect health by shaping the composition of the infant's gut microbiome. Breast milk oligosaccharides (HMOs) are the main soluble solids in breast milk, with 2 '-fucosyllactose (2' -FL) being the most abundant HMOs, accounting for 30% of the total. It is composed of L-fucose, D-galactose and D-glucose. The biological activity of 2' -FL has attracted much attention in recent years and is widely recognized by researchers as an important factor in the health and development of infants. Intensive studies have found that infants, although having difficulty digesting 2' -FL, play a crucial role in the immune system in development, such as regulating the gut and inhibiting pathogen infection. Currently, 2' -FL has been approved by the us, europe etc. as a prebiotic for infant formula.
In recent years, researchers have begun to use microbial fermentation method to produce 2' -FL, mainly using Escherichia coli and yeast and other model microorganisms. Coli itself contains multiple genes of the 2' -FL synthesis pathway, such as mannomutase, mannose-1-phosphate-guanylyltransferase, etc., and it is easy to construct a complete synthesis pathway, and thus research is the most extensive. However, escherichia coli is not a food-safe microorganism, and the product produced therefrom has problems in application to food. Since Bacillus subtilis is a food-safe strain, researchers have utilized this host for the construction and fermentative production of the 2' -FL synthetic pathway. The main problems existing at present are that the expression quantity of heterologous genes in bacillus subtilis is low, so that the synthesis efficiency of the way is not high; on the other hand, in the existing 2' -FL fermentation, the bacillus subtilis mainly takes fine carbon sources such as glucose, glycerol and the like as a culture medium, but has poor effect when a corn flour raw material with wider source and lower price is used. In addition, both E.coli and B.subtilis are at relatively high risk of phage infection in large-scale production.
Bacillus licheniformis is a widely used as a production host for food enzyme preparations and important nutritional chemicals, and the product is approved by the FDA as "general regulated as safe" (GRAS) level. In industrial fermentation, the bacillus licheniformis can ferment more raw materials, and has stronger resistance to bacteriophage. The existing 2' -FL synthesis routes of strains all use glucose, fructose or glycerol as starting substrates. The bacillus licheniformis can secrete mannanase, so that the mannan outside the cell is decomposed for growth and metabolism. Since mannose is an important intermediate compound of the 2'-FL synthesis pathway, mannose directly decomposes extracellular mannan and transports the mannose into cells to participate in the synthesis of 2' -FL, the method has obvious advantages in terms of production efficiency and cost compared with the existing synthesis route.
Disclosure of Invention
The invention aims to provide a promoter capable of efficiently expressing a target gene in bacillus licheniformis and construction and application of a recombinant plasmid.
According to the invention, through metabolic engineering modification, the bacillus licheniformis can utilize the konjac flour which is a cheap raw material, decompose and efficiently transport mannose into cells, and synthesize GDP-fucose by taking the GDP-mannose as a precursor, and then heterologously express fucosyltransferase to obtain the bacillus licheniformis engineering strain with high 2' -FL yield. The artificial approach for synthesizing 2' -FL by bacillus licheniformis takes mannan substrates (konjak flour, guar gum and the like) and corn flour as carbon sources, and utilizes self-mannase and corn flour to perform enzymatic hydrolysis to obtain mannose and glucose. Glucose is used as an energy source of cells, mannose is phosphorylated by a phosphate transfer system, and then mannose hexaphosphate is obtained by using phosphomannose mutase expressed in a heterologous way. Then obtaining GDP-L-fucose by respectively carrying out heterogeneously expressed mannose-1-phosphate-guanylyltransferase, GDP-mannose-4,6-dehydratase and GDP-L-fucose synthetase, and obtaining 2' -FL by utilizing fucosyltransferase (figure 1).
The invention provides a high-2' -FL-yield Bacillus licheniformis engineering strain, wherein mannose-specific phosphotransferase (N1), phosphomannomutase (N2), mannose-1-phosphate-guanylyltransferase (N3), GDP-mannose-4,6-dehydratase (N4), GDP-L-fucose synthetase (N5) and fucosyltransferase (N6) are overexpressed in the Bacillus licheniformis engineering strain.
In one embodiment, the engineered strain of Bacillus licheniformis over-expresses an expression cassette comprising a mannose-specific phosphotransferase transporter gene, an expression cassette comprising a gene encoding a phosphomannomutase, an expression cassette comprising a gene encoding mannose-1-phosphoguanylate transferase, an expression cassette comprising a gene encoding GDP-mannose-4,6-dehydratase, an expression cassette comprising a gene encoding GDP-L-fucose synthase, and an expression cassette comprising a gene encoding fucosyltransferase.
In one embodiment, the mannose-specific phosphotransferase transporter protein is derived from Escherichia coli, phosphomannomutase is derived from Escherichia coli, mannose-1-phosphate-guanyltransferase is derived from Escherichia coli, GDP-mannose-4,6-dehydratase is derived from Escherichia coli, GDP-L-fucose synthase is derived from Escherichia coli, and fucosyltransferase is derived from helicobacter pylori.
In one embodiment, a promoter P is utilized lan The gene is overexpressed, and the expression of the gene is terminated by using a terminator ter.
In one embodiment, the amino acid sequence of the mannose-specific phosphotransfer transporter is set forth in SEQ ID No. 3; the amino acid sequence of the phosphomannose mutase is shown as SEQ ID NO. 4; the amino acid sequence of the mannose-1-phosphate-guanylyltransferase is shown as SEQ ID NO. 5; the amino acid sequence of the GDP-mannose-4,6-dehydratase is shown in SEQ ID NO. 6; the amino acid sequence of the GDP-L-fucose synthetase is shown in SEQ ID NO. 7; the amino acid sequence of the fucosyltransferase is shown in SEQ ID NO. 8.
In one embodiment, the starting strain of the engineered strain of Bacillus licheniformis is Bacillus licheniformis ATCC9945A.
In one embodiment, the above engineered Bacillus licheniformis bacteria uses plasmids pHY and pHT43 as expression vectors.
In one embodiment, the mannose-specific phosphotransferase transporter, phosphomannomutase, and mannose-1-phosphate-guanylyltransferase are expressed using plasmid pHY, and GDP-mannose-4,6-dehydratase, GDP-L-fucose synthase, and fucosyltransferase are expressed using plasmid pHT 43.
In one embodiment, the nucleotide sequence of the gene encoding the mannose-specific phosphotransferase transporter is set forth in SEQ ID
No.9, the nucleotide sequence of the gene encoding phosphomannose mutase is shown in SEQ ID No.10, and the gene encoding mannose
The nucleotide sequence of the-1-phospho-guanylate transferase gene is shown in SEQ ID No.11, the nucleotide sequence of the gene coding GDP-mannose-4,6-dehydratase is shown in SEQ ID No.12, the nucleotide sequence of the gene coding GDP-L-fucose synthetase is shown in SEQ ID No.13, and the nucleotide sequence of the gene coding fucosyltransferase is shown in SEQ ID No. 14.
In one embodiment, the promoter P lan The nucleotide sequence of (A) is shown in SEQ ID NO.1, and the nucleotide sequence of terminator ter is shown in SEQ ID NO. 2.
The invention also provides a construction method of the bacillus licheniformis for efficiently synthesizing 2' -fucosyllactose, wherein the bacillus licheniformis contains plasmids pHY and pHT43; the plasmid pHY contains a gene for coding mannose-specific phosphotransferase transporter, a gene for coding phosphomannose mutase and a gene for coding mannose-1-phosphate-guanylyltransferase, and the plasmid pHT43 contains a gene for coding GDP-mannose-4,6-dehydratase, a gene for coding GDP-L-fucose synthetase and a gene for coding fucosyltransferase.
In one embodiment, a promoter P is utilized lan The gene is overexpressed, and the expression of the gene is terminated using a terminator ter.
In one embodiment, the nucleotide sequence of the gene encoding the mannose-specific phosphotransfer transporter is set forth in SEQ ID
No.9, the nucleotide sequence of the gene encoding phosphomannose mutase is shown in SEQ ID No.10, and the gene encoding mannose
The nucleotide sequence of the-1-phosphate-guanylate transferase gene is shown as SEQ ID NO.11, the nucleotide sequence of the gene coding GDP-mannose-4,6-dehydratase is shown as SEQ ID NO.12, the nucleotide sequence of the gene coding GDP-L-fucose synthetase is shown as SEQ ID NO.13, and the nucleotide sequence of the gene coding fucosyltransferase is shown as SEQ ID NO. 14.
In one embodiment, the promoter P lan The nucleotide sequence of (A) is shown in SEQ ID NO.1, and the nucleotide sequence of terminator ter is shown in SEQ ID NO. 2.
In one embodiment, the construction method comprises the steps of:
(1) pHY is used as starting plasmid, and promoter P with quorum sensing effect is used lan Overexpression of mannose-specific phosphotransfer transporter gene on plasmid pHY, and transformation of Escherichia coli with the constructed recombinant plasmid to obtain plasmid pHY-N1;
(2) pHY-N1 is used as starting plasmid, into which promoter P is inserted lan The expression frame of the phosphomannose mutase coding gene and the terminator, and transforming the constructed recombinant plasmid into escherichia coli to obtain a plasmid pHY-N12;
(3) pHY-N12 is used as starting plasmid, into which promoter P is inserted lan The mannose-1-phosphate-guanylyltransferase coding gene and the expression frame of the terminator ter, and the constructed recombinant plasmid is transformed into escherichia coli to obtain a plasmid pHY-N123;
(4) Starting from pHT43A particle into which a promoter P is inserted lan GDP-mannose-4,6-dehydratase coding gene and terminator ter expression frame, and transforming the constructed recombinant plasmid into escherichia coli to obtain plasmid pHT-N4;
(5) pHT43-N4 is used as starting plasmid, into which promoter P is inserted lan GDP-L-fucose synthetase coding gene and terminator ter, and the constructed recombinant plasmid is transformed into escherichia coli to obtain plasmid pHT-N45;
(6) pHT43-N45 is used as starting plasmid, into which P containing promoter is inserted lan The fucosyltransferase coding gene and the expression frame of terminator ter, and the constructed recombinant plasmid is transformed into escherichia coli to obtain the plasmid pHT-N456.
(7) The plasmid pHY-N123 is transformed into Bacillus licheniformis ATCC9945A to obtain recombinant strain BLH5.
(8) And transforming the plasmid pHT-N456 into Bacillus licheniformis BLH5 to obtain recombinant strain BLH6.
The invention provides a method for producing 2 '-fucosyllactose, which is to use the bacillus licheniformis engineering strain to produce 2' -fucosyllactose by fermentation in a fermentation system taking mannan as a carbon source.
In one embodiment, the mannan comprises konjac flour, guar gum.
In one embodiment, the fermentation system further comprises corn flour and lactose.
In one embodiment, the seed solution of the bacillus licheniformis engineering bacteria is inoculated into a fermentation system, the air flow is controlled to be 0.5vvm, the stirring and DO coupling is controlled to be 25-35%, the rotating speed is set to be 200-800 rpm, and 60-80 g/L corn flour and 60-80 g/L konjac flour are supplemented once after fermentation is carried out for 10-14 hours.
In one embodiment, the seed solution is prepared by streaking the above engineered Bacillus licheniformis bacteria on a plate, selecting a single colony, inoculating to LB medium, culturing at 37 deg.C and 250rpm/min for 18-24 h, inoculating 2ml of the bacterial solution to 100ml of LB liquid medium, culturing at 37 deg.C and 250rpm for 18-24 h.
The invention also provides application of the bacillus licheniformis in synthesizing 2 '-fucosyllactose or products containing 2' -fucosyllactose.
Compared with the prior art, the invention has the positive improvement effects that:
according to the invention, the bacillus licheniformis is used as an original strain, and by utilizing the characteristic that the bacillus licheniformis can secrete mannanase, the mannose transport capacity is enhanced, so that the strain can directly and efficiently obtain mannose from the outside of cells and participate in a 2' -FL synthesis route. On the other hand, the promoter P with quorum sensing effect is adopted lan Expressing mannose-specific phosphotransferase transporter, phosphomannomutase, mannose-1-phosphate-guanylyltransferase, GDP-mannose-4,6-dehydratase, GDP-L-fucose synthetase and fucosyltransferase, coordinating cell growth and product synthesis, increasing the yield and productivity of 2'-FL, increasing the yield of 2' -FL to 98g/L, and increasing the maximum OD 600 Is 63. Bacillus licheniformis is a food safety type industrial microorganism, and cheap mannan raw materials such as konjac flour and the like are used as a carbon source to ferment and produce 2' -FL; compared with escherichia coli and bacillus subtilis which utilize fine carbon sources such as glucose or glycerol, the technical scheme of the invention has remarkable advantages in terms of production cost and efficiency.
Drawings
FIG. 1: design of Bacillus licheniformis 2' -FL synthetic pathway
FIG. 2: structure diagram of recombinant plasmid; a, pHYN123 expression vector, B, pHTN456 expression vector;
FIG. 3: physical map of recombinant plasmid;
FIG. 4: chromatographic quantitative analysis of 2' -FL in the fermentation supernatant;
FIG. 5: the content of 2' -FL in the shake flask changes with time under different culture temperature conditions;
FIG. 6: the process curve of the recombinant bacterium fed-batch fermentation production of 2' -FL.
Detailed Description
The present invention will be described in detail with reference to examples for better understanding the objects, technical solutions and advantages of the present invention, but they should not be construed as limiting the scope of the present invention.
(I) culture Medium
LB culture medium: 10g/L of peptone, 5g/L of yeast powder and 10g/L of sodium chloride; 20g/L agar powder was added to prepare an LB solid medium.
Shake flask fermentation medium: 75g/L of sucrose, 40g/L of lactose and 30g/L, K of cottonseed protein 2 HPO 4 ·3H 2 O 9.12g/L、KH 2 PO 4 1.36 g/L、(NH 4 ) 2 HPO 4 10g/L; initial pH7.5.
Fermentation medium: 30g/L of cottonseed protein, 75g/L of konjac flour, 75g/L of corn flour and 80g/L, K of lactose 2 HPO4·3H 2 O9.12g/L、KH 2 PO 4 1.36 g/L、FeCl 3 0.5 g/L、(NH 4 ) 2 HPO 4 10 g/L(pH 7.5)。
Extraction and detection of (di) 2' -FL
The fermentation liquor is centrifuged at 12000r/min for 5min, and the supernatant is filtered by a 0.22 μm membrane and detected by HPLC. HPLC detection conditions: a differential refractive detector; the chromatographic column is Polyamino HILIC 5 μm 250 x 4.6mm (Dima science and technology, china), and the column temperature is 40 deg.C; the mobile phase is 75% acetonitrile water solution, and the flow rate is 0.8mL/min; the sample size was 10. Mu.L.
(III) strains and primers referred to in the following examples.
TABLE 1 strains involved in the invention
TABLE 2 primer sequences
Example 1 construction of recombinant Bacillus licheniformis BLH6
PCR of P by overlap extension lan The promoter (shown in a nucleotide sequence SEQ ID NO. 1) and the terminator of xylose isomerase gene (shown in a nucleotide sequence SEQ ID NO. 2) are respectively fused with gene segments of mannose-specific phosphotransferase transporter (N1), phosphomannose mutase (N2), mannose-1-phosphate-guanyltransferase (N3), GDP-mannose-4,6-dehydratase (N4), GDP-L-fucose synthetase (N5) and fucosyltransferase (N6) to obtain gene expression segments SEQ ID NO.9-14.
The primer pairs P1-P6 are respectively used for amplifying gene expression fragments SEQ ID NO.9-14, so that enzyme cutting sites are introduced at two ends of the gene fragments. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, then entering next cycle of denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 40s, and 30 cycles; extension at 72 ℃ for 10min and incubation at 4 ℃.
The expression cassette containing N1, N2 and N3 was inserted into the plasmid by homologous recombination using restriction sites HindIII, bglII, bglII and SalI (for ligation to pHY plasmid) in this order to obtain pHY-N123 (FIG. 2). The expression cassettes containing N4, N5, N6 were inserted into the plasmid by homologous recombination using restriction sites EcoRI, bamHI, smaI and SacI in order (for ligation of pHT43 plasmid), giving pHT-N456 (FIG. 2). One gene is inserted between every two adjacent enzyme cutting sites. The plasmid construction was further verified by 1% agarose gel electrophoresis (FIG. 3).
The successfully constructed recombinant plasmid pHY-N123 is subjected to the following steps of Li, Y; jin, k.; zhang, l.; ding, z.; gu, z.; the method of Shi, G.development of an induced Expression System in Bacillus licheniformis Based on an Engineered Xylose operon. Journal of agricultural and Food Chemistry 2018,66,9456-9464 was introduced into Bacillus licheniformis ATCC9945A to obtain recombinant Bacillus licheniformis BLH5.
The successfully constructed recombinant plasmid pHT-N456 was introduced into BLH5 in the same manner as described above to obtain recombinant Bacillus licheniformis BLH6.
EXAMPLE 2 Shake flask fermentation of 2' -FL
The recombinant strain BLH6 constructed in example 1 was streaked and activated on LB plate, cultured at 37 ℃ for 16 hours, and then single colony was selected and inoculated in 15mL LB medium at 37 ℃ and 250 r.min -1 Culturing for 16-18 h to obtain seed solution, transferring 1mL seed solution into 30mL shake flask fermentation medium with initial OD of 0.5-1 at 37 deg.C or 42 deg.C and 250r min -1 And (5) culturing. Sampling every 12h, fermenting at 4 deg.C at 12000 r.min -1 Centrifugation was carried out for 10min under these conditions and the supernatant was used for detection of the product (FIG. 4). As shown in FIG. 5, 1.82g/L of 2'-FL was accumulated in the 36-hr fermentation broth under the culture condition at 42 ℃ and the yield of 2' -FL in the 36-hr fermentation broth was 1.52g/L at 37 ℃.
Example 3 Effect of carbon Source type and concentration on recombinant expression
The recombinant strain BLH6 constructed in example 1 was streaked and activated on LB plate, cultured at 37 ℃ for 16 hours, and then single colony was selected and inoculated in 15mL LB medium at 37 ℃ and 250 r.min -1 Culturing for 16-18 h to obtain seed solution, transferring 1mL seed solution into 30mL shake flask fermentation medium with initial OD of 0.5-1 at 37 deg.C or 42 deg.C and 250r min -1 And (5) culturing. Fermenting for 36h, fermenting at 4 deg.C for 12000r min -1 Centrifuging for 10min under the condition, and using the supernatant for detecting the product.
The carbon source in the shake flask fermentation medium is replaced by konjac flour or guar gum with different concentrations (the concentrations are respectively 30g/L, 45g/L, 60g/L, 75g/L and 90 g/L) so as to explore the influence of different carbon source types and concentrations on the fermentation production of 2' -FL by recombinant BLH6. As shown in Table 3, the maximum yield of 2' -FL was 11.32g/L using 75g/L of konjac flour as a carbon source; and 5.74 g/L2' -FL can be obtained by using 75g/L guar gum as a carbon source. This indicates that the recombinant Bacillus licheniformis can grow and synthesize the product better in different culture mediums which use cheap mannan raw materials as main carbon sources.
TABLE 3 influence of different carbon source types and concentrations on the fermentation of recombinant bacteria
Example 4 production of 2' -FL at 20L fermenter Scale
The recombinant strain BLH6 constructed in example 1 was streaked and activated on LB plate, cultured at 37 ℃ for 16 hours, and then single colony was selected and inoculated in 15mL LB medium at 37 ℃ and 250 r.min -1 Culturing for 18-24 h to obtain primary seed solution, transferring 2mL of the primary seed solution into 100mL LB medium at 37 deg.C for 250r min -1 Culturing for 18-24 h to obtain second-stage seed solution, inoculating 3 bottles of 2-stage seed solution into 30L fermentation tank containing 15L fermentation medium, and fermenting.
The fermentation temperature was 42 ℃ and the initial pH was 7.5. When the pH value is reduced to 7.0 in the fermentation process, 50% ammonia water is supplemented to maintain the pH value at about 7.0 in the fermentation process, the ventilation amount is controlled at 0.5vvm in the fermentation process, the stirring and DO coupling control DO is maintained at about 30%, and the upper limit of the rotation speed is set to 800rpm. After 12 hours of fermentation, 75g/L corn flour and 75g/L konjac flour are supplemented once.
The fermenter-scale fed-batch fermentation results are shown in FIG. 6, where the yield of 2' -FL reached 98g/L and the maximum OD reached after 46h of fermentation 600 Is 63.
TABLE 4 fermenter Process parameters
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A bacillus licheniformis engineering strain with high 2' -FL yield is characterized in that mannose-specific phosphotransferase transporter, phosphomannomutase, mannose-1-phosphate-guanylyltransferase, GDP-mannose-4,6-dehydratase, GDP-L-fucose synthetase and fucosyltransferase are overexpressed in the bacillus licheniformis engineering strain.
2. The engineered Bacillus licheniformis strain of claim 1, wherein the mannose-specific phosphotransferase transporter is derived from Escherichia coli, phosphomannomutase is derived from Escherichia coli, mannose-1-phosphate-guanyltransferase is derived from Escherichia coli, GDP-mannose-4,6-dehydratase is derived from Escherichia coli, GDP-L-fucose synthase is derived from Escherichia coli, and fucosyltransferase is derived from helicobacter pylori.
3. The engineered Bacillus licheniformis strain according to claim 1, wherein the promoter P is used lan The gene is overexpressed, and the expression of the gene is terminated using a terminator ter.
4. The engineered bacillus licheniformis strain of claim 1, wherein the starting strain of the engineered bacillus licheniformis strain is bacillus licheniformis ATCC9945A, and the plasmids pHY and pHT43 are expression vectors.
5. The engineered Bacillus licheniformis strain of claim 4, wherein the mannose-specific phosphotransferase transporter, phosphomannomutase and mannose-1-phosphate-guanylate transferase are expressed from plasmid pHY and the GDP-mannose-4,6-dehydratase, GDP-L-fucose synthetase and fucosyltransferase are expressed from plasmid pHT 43.
6. A construction method of Bacillus licheniformis for efficiently synthesizing 2' -fucosyllactose is characterized in that the Bacillus licheniformis contains plasmids pHY and pHT43; the plasmid pHY contains a gene for coding mannose-specific phosphotransferase transporter, a gene for coding phosphomannomutase and a gene for coding mannose-1-phosphate-guanylyltransferase, and the plasmid pHT43 contains a gene for coding GDP-mannose-4,6-dehydratase, a gene for coding GDP-L-fucose synthetase and a gene for coding fucosyltransferase.
7. A method for producing 2 '-fucosyllactose, which is characterized in that the method uses the bacillus licheniformis engineering strain as described in any claim 1-5 as a fermentation strain, and produces 2' -fucosyllactose by fermentation in a fermentation system using mannan as a carbon source.
8. The method of claim 7, wherein the mannan comprises konjac flour, guar gum.
9. The method of claim 7, wherein the fermentation system further comprises corn flour and lactose.
10. Use of a bacillus licheniformis according to any of the claims 1-5 for the synthesis of 2 '-fucosyllactose or products containing 2' -fucosyllactose.
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