CN117025650A - Recombinant E.coli for producing complex fucosylated lactose - Google Patents

Recombinant E.coli for producing complex fucosylated lactose Download PDF

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CN117025650A
CN117025650A CN202311080390.3A CN202311080390A CN117025650A CN 117025650 A CN117025650 A CN 117025650A CN 202311080390 A CN202311080390 A CN 202311080390A CN 117025650 A CN117025650 A CN 117025650A
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fucosyllactose
fut
expression
lactose
lacy
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周洪波
刘雯娴
王玉光
陈祝
程海娜
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Central South University
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Central South University
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Abstract

The invention relates to the field of microbial genetic engineering, in particular to recombinant escherichia coli for producing composite fucosylated lactose. The invention adopts escherichia coli as chassis cells to produce composite fucosyl lactose, and realizes the simultaneous production of three fucosyl lactose by regulating and controlling the expression levels of 11 genes or enzymes participating in the synthesis paths of 2' -fucosyl lactose, 3-fucosyl lactose and difucosyl lactose. The preparation process of the composite fucosylated lactose provided by the invention is simple, the cost is low, the efficiency is higher, and meanwhile, the composite fucosylated lactose with different mass compositions can be obtained through the expression level regulation and control of different genes or enzymes.

Description

Recombinant E.coli for producing complex fucosylated lactose
Technical Field
The invention relates to the field of microbial genetic engineering, in particular to recombinant escherichia coli for producing composite fucosylated lactose.
Background
Breast milk is the gold standard of infant formula milk powder. Only the opening of the mystery of breast milk has led to a breakthrough in the infant food industry. Breast milk oligosaccharides (HumanMilk Oligosaccharides, HMOs) are an important component of breast milk, and are present in amounts inferior to lactose. As a prebiotic, the microbial agent can promote the colonization of the intestinal tracts of infants, can enhance the immunity of infants, is the most important guarantee for resisting the invasion of external bacteria and viruses, plays a key role in early life health of infants, and has important research significance and development value.
Fucosylated oligosaccharides are the highest-content oligosaccharides in HMOs, accounting for more than 70%, and have been widely used in the food and pharmaceutical field, especially for addition to infant formulas. The biosynthesis process can be understood as (for example two oligosaccharides): FUT2 (α -1, 2-fucosyltransferase) adds Fuc (fucosyl) at the terminal Gal (galactosyl) via an α -1-2 linkage; FUT3 (α -1, 3-fucosyltransferase) adds Fuc (fucosyl) to the internal GlcNAc (acetamido-glucosyl) via the α -1-4 chain. They belong to the same family as fucosyltransferases and are involved in the synthesis of fucosylated oligosaccharides. The catalytic characteristic is that it is capable of transferring nucleotide-activated fucosyl groups onto various sugar receptors. Fucosylation is generally the last step in the biosynthesis of such oligosaccharides. Thus, the type of fucosyltransferase determines the type of oligosaccharide.
2 '-fucosyllactose (2' -FL), 3-fucosyllactose (3-FL) and Difucosyllactose (DiFL) are natural oligosaccharides derived from breast milk, with lactose as the basic sugar unit. 2 '-fucosyllactose (2' -FL) and 3-fucosyllactose (3-FL) differ in that the fucosyl groups are on different monosaccharide glycosyl units; difucosyllactose (DiFL) is a compound in which one fucosyl group is added to both glycosyl units. Breast milk oligosaccharides are usually present in complex form of multiple components.
In the prior art, there is no cell factory production strategy for simultaneously achieving multiple fucosylated oligosaccharides. However, many reports have been made on the construction and use of single fucosylated oligosaccharides such as 2 '-fucosyllactose (2' -FL) and 3-fucosyllactose (3-FL) in cell factories. Wherein, chassis cells such as corynebacterium glutamicum, escherichia coli cells and the like are mostly adopted to produce single products such as 2' -fucosyllactose and the like, a plurality of genes are mostly changed in engineering strains, disturbance and burden on basic growth metabolism of cells are probably larger, hidden dangers which are unknown are probably caused for industrial production, and one-step synthesis of a plurality of products cannot be realized.
Therefore, when 2 '-fucosyllactose (2' -FL), 3-fucosyllactose (3-FL) and Difucosyllactose (DiFL) are industrially produced, production by one means that the above three products need to be fermented, extracted and purified separately and then re-compounded into HMO products. Such a process is complicated, and the cost increases, which in turn reduces the efficiency.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide recombinant escherichia coli producing complex fucosylated lactose, and the present invention provides recombinant strain producing complex fucosylated lactose, which not only can satisfy the production of a single product, but also can realize one-step synthesis of various HMO products through further modification.
The present invention provides the use of modulating the expression level of one or more of manA, cpsG, cpsB, gmd, fcl, galU, galE, alpha-1, 2-Fut, alpha-1, 3-Fut, lacY and SetA to increase the yield of 2' -fucosyllactose, 3-fucosyllactose and/or difucosyllactose.
Compared with the prior art, the method realizes the simultaneous production of three kinds of fucosyllactose by regulating and controlling the expression levels of 11 genes or enzymes participating in the synthesis paths of 2 '-fucosyllactose, 3-fucosyllactose and difucosyllactose, and further shortens the fermentation period of 2' -fucosyllactose and 3-fucosyllactose, thereby realizing more accurate technical effects.
In some embodiments, the modulation comprises at least one of the following (1) - (5):
(1) increasing the level of gene expression by optimizing codons;
(2) adding molecular chaperones to increase the gene expression level;
(3) promoters or enhancers that enhance or modify gene expression increase the level of gene expression;
(4) improving the expression level of the gene by modifying the ribosome binding site of the gene;
(5) and adding an inducer for promoting gene expression to improve the gene expression level.
The invention provides a recombinant plasmid, which comprises a vector skeleton and at least one of manA, cpsG, cpsB, gmd, fcl, galU, galE, alpha-1, 2-Fut, alpha-1, 3-Fut, lacY and SetA.
In some embodiments, the vector backbone comprises a low copy expression plasmid and/or a high copy expression plasmid.
In some embodiments, the vector backbone includes nucleic acid sequences or elements necessary for expression in the strain, including promoters, ribosome binding sites, and possibly other sequences and elements. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself.
The low copy expression plasmid comprises pACYC plasmid, and the high copy expression plasmid comprises pET series plasmid.
In other embodiments, the present invention further provides a combination of strong and weak expression levels by modifying the promoters, ribosome binding sites and possibly other sequences and elements of the low-copy expression plasmid and the high-copy expression plasmid to regulate and control different genes to enhance expression levels, thereby achieving more excellent effects.
In some embodiments, the recombinant plasmid comprises at least one of the following (1) - (3):
(1) The manA, cpsG, cpsB, galU, galE, lacY and SetA ligate low copy expression plasmids, the gmd, fcl, α -1,2-Fut and α -1,3-Fut ligate high copy expression plasmids;
(2) The manA, cpsG, cpsB, galU, galE and α -1,3-Fut are linked to a low copy expression plasmid, and the gmd, fcl, α -1,2-Fut, lacY and SetA are linked to a high copy expression plasmid;
(3) The manA, cpsG, cpsB, galU, galE and alpha-1, 2-Fut were ligated to low copy expression plasmids, and the gmd, fcl, alpha-1, 3-Fut, lacY and SetA were ligated to high copy expression plasmids.
In some embodiments, one or more genes may be carried simultaneously on one plasmid. The different plasmids only differ in the different antibiotic species, which ensures that they have the same copy number and are thus controlled to the same expression intensity.
The invention provides a genetic engineering strain for producing 2' -fucosyllactose, 3-fucosyllactose and/or difucosyllactose, wherein the expression level of at least one gene of manA, cpsG, cpsB, gmd, fcl, galU, galE, alpha-1, 2-Fut, alpha-1, 3-Fut, lacY and SetA in the genetic engineering strain is up-regulated.
In some specific embodiments, the expression levels of manA, cpsG, cpsB, gmd, fcl, galU, galE, alpha-1, 2-Fut, alpha-1, 3-Fut, lacY and SetA in the genetically engineered strain are up-regulated, so that simultaneous production of 2' -fucosyllactose, 3-fucosyllactose and difucosyllactose is realized, and a better technical effect is obtained.
The invention provides a construction method of the genetic engineering strain, which comprises the steps of transforming or transfecting the recombinant plasmid into a host strain.
Host strains according to the invention, including fungi, bacteria and algae. In some embodiments, the host strain comprises escherichia coli.
In some embodiments, the E.coli is E.coli W3110 (DE 3).
The invention provides a preparation method of 2 '-fucosyllactose, 3-fucosyllactose and/or difucosyllactose, which comprises culturing the genetic engineering strain or the genetic engineering strain obtained by the construction method to obtain a culture containing the 2' -fucosyllactose, 3-fucosyllactose and/or difucosyllactose.
In some embodiments, the medium of the culturing includes glucose and/or lactose.
In some embodiments, the culturing comprises fermenter culturing.
The invention provides application of 2' -fucosyllactose, 3-fucosyllactose and/or difucosyllactose prepared by the preparation method in preparation of products for improving intestinal immunity.
The invention provides a product for improving intestinal immunity, which comprises at least one of the following raw materials 1) to 4):
1) The recombinant plasmid;
2) The genetically engineered strain;
3) Genetically engineered strain obtained by the construction method
4) The culture prepared by the preparation method.
The invention provides recombinant E.coli producing complex fucosylated lactose. The invention adopts escherichia coli as chassis cells, and realizes the simultaneous production of three fucosyllactoses by regulating and controlling the expression levels of 11 genes or enzymes participating in the synthesis paths of 2' -fucosyllactose, 3-fucosyllactose and difucosyllactose. The preparation process of the composite fucosylated lactose provided by the invention is simple, the cost is low, the efficiency is higher, and meanwhile, the composite fucosylated lactose with different mass compositions can be obtained through the expression level regulation and control of different genes or enzymes. Compared with the prior art, the fermentation period of the 2 '-fucosyllactose and the 3-fucosyllactose is obviously shortened, the yield of the 2' -fucosyllactose within 48 hours is 3.2g/L, and the yield of the 3-fucosyllactose is 1.4g/L, which are higher than those of the prior art, and the invention has industrial application prospect.
Drawings
FIG. 1 shows a schematic diagram of the synthetic pathway of complex fucosyllactose in the examples;
FIG. 2 is a graph showing the results of shake flask fermentation verification of the optimized strain 4 in the effect example.
Detailed Description
The invention provides recombinant escherichia coli for producing complex fucosylated lactose, and a person skilled in the art can refer to the content of the invention and appropriately improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples.
EXAMPLE 1 preparation of Complex fucosyllactose
In the scheme, escherichia coli is selected as a chassis cell, and the synthesis flux of nucleotide activated fucose is enhanced by optimizing the original anabolic pathway of the cell, so that a cell factory suitable for producing fucosylated lactose is obtained. Then, integrating alpha-1, 2-fucosyltransferase to complete the synthesis of 2' -fucosyllactose; integrating alpha-1, 3-fucosyltransferase to complete synthesis of 3-fucosyllactose; the synthesis of 2' -fucosyllactose, 3-fucosyllactose and difucosyllactose is accomplished by integrating alpha-1, 2-fucosyltransferase and alpha-1, 3-fucosyltransferase.
In more detail, we divide the fucosyllactose synthesis into three parts: the precursor synthesis portion, the product formation portion, and the product discharge portion, and the specific synthesis pathways are shown in FIG. 1. The scheme has innovative work in all parts.
Precursor synthesis part: coli W3110 was selected as the starting strain. No heterologous gene is required to be expressed under this protocol. By modulating the different expression levels of key genes in the precursor synthesis pathway, a significant increase in the flux of fucosylated fucose synthesis can be achieved.
Product formation (red circle in fig. 1): the synthesis of 2' -fucosyllactose, 3-fucosyllactose and difucosyllactose can be accomplished by expressing the alpha-1, 2-fucosyltransferase, the alpha-1, 3-fucosyltransferase and the two enzymes together, respectively. This is a new work to our knowledge. Here, when the alpha-1, 2-fucosyltransferase and the alpha-1, 3-fucosyltransferase are co-expressed, the product theoretically contains only 2' -fucosyllactose and 3-fucosyllactose. However, as the fucosylation process proceeds, fucosyl groups are added to the fucosylated glycosyl sites, thereby producing a difucosylated lactose. This means that the gene expression level and catalytic efficiency of two key synthetases have a significant impact on the ratio of product compositions. In this protocol we found its optimal level of gene expression.
Product discharge section (grey column in fig. 1): under the scheme, the engineering strain only needs lactose as a single substrate except a basal culture medium for self growth and reproduction. Wherein, engineering strains need to efficiently absorb lactose into cells, and simultaneously, high efficiency discharge products are needed to promote continuous synthesis reaction. It is therefore necessary to express two key proteins of interest to accomplish the relevant function.
As shown in FIG. 1, related genes involved in fucosylation oligosaccharide biosynthesis are modified, namely, expression levels of manA (JW 1605), cpsG (JW 2033), cpsB (JW 2034), gmd (JW 2038), fcl (JW 2037), galU (JW 1224) and galE (JW 0742) genes are regulated in a genome or plasmid overexpression mode, and carbon fluxes of anabolic pathways are precisely regulated through combination of different gene intensities. Then, we integrated different fucosyltransferases to achieve the synthesis of different fucosylated lactose products. By expressing LacY (JW 0334) and SetA (JW 0069), the pressure of substrate entry and exit in the cell factory is dredged, further improving the efficiency of product synthesis.
Coli W3110 (DE 3) was selected as the basal cell. The corresponding primers were designed to amplify and purify the target gene sequence, which was then ligated to the pET series plasmid. One plasmid may carry one or more genes simultaneously. The different plasmids only differ in the different antibiotic species, which ensures that they have the same copy number and are thus controlled to the same expression intensity. The successfully constructed plasmid was transformed into E.coli W3110 (DE 3). Screening to obtain recombinant strain containing plasmid.
As shown in Table 1, the introduction of exogenous alpha-1, 2-fucosyltransferase and alpha-1, 3-fucosyltransferase alone into a strain hardly caused the strain to produce the desired HMOs oligosaccharides. Whereas higher titers of 2' -FL and 3-FL compared to the wild-type can be obtained in the presence of alpha-1, 2-fucosyltransferase and alpha-1, 3-fucosyltransferase after conventional overexpression of manA, cpsG, cpsB, gmd, fcl, galU, galE. Whereas when both α -1, 2-fucosyltransferase and α -1, 3-fucosyltransferase are expressed and the transporters LacY and SetA are overexpressed, the titres of 2' -FL and 3-FL are slightly decreased, but the synthesis of the third HMOs oligosaccharide, diFL, is promoted at this time.
TABLE 1
Description: fermenting for 24h; not optimized, +: up-regulation of expression.
The yield of the target fucosylated lactose can be further increased by optimizing the expression levels of different genes and enzymes in the synthetic pathway of FIG. 1, resulting in 5 optimized strains, the specific optimization process and results are shown in Table 2.
TABLE 2
Description: fermenting for 48h; low copy plasmid, ++: high copy plasmid,/: not detected.
In order to further explore the influence of different gene expression doses on target yield synthesis, different genes are expressed through plasmids with different copy numbers, so that the regulation and control of the different gene doses are realized.
In optimized strain 1, low copy plasmids such as pACYC were used to carry manA, cpsG, cpsB, galU, galE, lacY and SetA, and high copy plasmids such as pET were used to carry gmd, fcl and α -1,2-Fut. The results indicate that it gives a higher 2' -FL titre relative to mutant 1.
Similarly, optimized strains 2 and 3 also exhibited higher product titers than mutant strains 2 and 3. This suggests that there is some subtle coordination between genes on the anabolic pathway. Excessive pursuit of high-level expression of genes may not achieve optimal results. And the enhancement of the expression level of different genes is carried out, so that the combination of the intensity on a certain expression level is realized, and the optimal solution can be obtained.
Thus, optimizing strains 4 and 5 reduced the gene expression levels of α -1, 3-fucosyltransferase and α -1, 2-fucosyltransferase, respectively, on the basis of the previous generations of strains, while increasing the gene doses of the transporters LacY and SetA. The results show that the product content ratios of 2' -FL, 3-FL and DiFL can be adjusted by regulating the expression level of fucosyltransferase. The expression level of alpha-1, 2-fucosyltransferase has a more pronounced effect on the yield of DiFL.
Compared with the prior art that the yield of 2 '-fucosyllactose is 3.81g/L after culturing for 70h (shake flask level), the fermentation period of the 2' -fucosyllactose is obviously shortened, the yield of 2 '-fucosyllactose within 48h is 3.2g/L, and the yield of 2' -fucosyllactose (g/L.h) is higher than that of the prior art, thus having industrial application prospect.
Compared with the yield of the 3-fucosyllactose of 2.01g/L (shake flask level) after 72h culture and culture in the prior art, the fermentation period of the 3-fucosyllactose is obviously shortened, the yield of the 3-fucosyllactose within 48h is 1.4g/L, and the yield (g/L.h) of the 3-fucosyllactose is higher than that of the prior art, so that the method has industrial application prospect.
The prior art has not yet reported the simultaneous production of 2' -fucosyllactose, 3-fucosyllactose and difucosyllactose.
The scheme is carried out in an escherichia coli K12 strain, and the related genes and enzyme proteins are derived from the K12 strain. The genes and enzyme proteins involved may be of different names but have the same function. Therefore, the purpose of the invention can be achieved by changing the strain and applying the same technical means in the scheme.
Due to the complexity of biological metabolism, the optimal solution under this scheme may be the highest level that can be achieved under current knowledge. However, with the discovery of new synthetic pathways and enzymes, there may be a correlation with the anabolic pathways involved in the present solution, and then the relevant regulation may be performed, so that the effect under the present invention may be even better.
The present invention is a yield verification performed at the shake flask level that if placed in a bioreactor, yield may be higher than existing levels even with the same strain and the same culture conditions. Alternatively, it was verified in the bioreactor that the same effect could be achieved even better using a strain that is not optimal in the present invention.
Effect example
Shake flask fermentation verification of optimized strain 4. The fermentation time is 48 hours, and the culture medium comprises the following components: 11.8g/L tryptone, 23.6g/L yeast extract, 9.4g/L K 2 HPO 4 KH of 2.2g/L 2 PO 4 4ml/L glycerol. The results of the statistics of fermentation time and yields of 2' -FL, 3-FL and DiFL are shown in FIG. 2.
The results indicated that 2' -FL was always the highest yield of product. The pattern of accumulation of 3-FL is consistent with 2' -FL, except for a slightly lower titer. In contrast, diffl begins synthesis after 12 hours. This suggests that there may be some competing relationship with the synthesis of 2' -FL and 3-FL. When the fermentation is completed for 48 hours, the content of 2' -FL accounts for more than half of the total HMOs species produced at this time. This result shows that the content control of 2' -FL, 3-FL and DiFL can be further achieved by fermentation process control. The method can greatly save time, space and cost from single variety of HMOs production to separation and purification to compounding process, and has great application value. This strain is most preferred for all products requiring HMOs compounding.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (10)

1. Use of modulating the expression level of one or more of manA, cpsG, cpsB, gmd, fcl, galU, galE, α -1,2-Fut, α -1,3-Fut, lacY and SetA to increase the yield of 2' -fucosyllactose, 3-fucosyllactose and/or difucosyllactose.
2. The use according to claim 1, wherein the regulation comprises at least one of the following (1) to (5):
(1) increasing the level of gene expression by optimizing codons;
(2) adding molecular chaperones to increase the gene expression level;
(3) promoters or enhancers that enhance or modify gene expression increase the level of gene expression;
(4) improving the expression level of the gene by modifying the ribosome binding site of the gene;
(5) and adding an inducer for promoting gene expression to improve the gene expression level.
3. The recombinant plasmid is characterized by comprising at least one of a vector skeleton manA, cpsG, cpsB, gmd, fcl, galU, galE, alpha-1, 2-Fut, alpha-1, 3-Fut, lacY and SetA.
4. The recombinant plasmid according to claim 3, wherein the vector backbone comprises a low copy expression plasmid and/or a high copy expression plasmid.
5. The recombinant plasmid according to claim 3 or 4, characterized in that it comprises at least one of the following (1) to (3):
(1) The manA, cpsG, cpsB, galU, galE, lacY and SetA ligate low copy expression plasmids, the gmd, fcl, α -1,2-Fut and α -1,3-Fut ligate high copy expression plasmids;
(2) The manA, cpsG, cpsB, galU, galE and α -1,3-Fut are linked to a low copy expression plasmid, and the gmd, fcl, α -1,2-Fut, lacY and SetA are linked to a high copy expression plasmid;
(3) The manA, cpsG, cpsB, galU, galE and alpha-1, 2-Fut were ligated to low copy expression plasmids, and the gmd, fcl, alpha-1, 3-Fut, lacY and SetA were ligated to high copy expression plasmids.
6. A genetically engineered strain producing 2' -fucosyllactose, 3-fucosyllactose and/or difucosyllactose, characterized in that the expression level of at least one of manA, cpsG, cpsB, gmd, fcl, galU, galE, α -1,2-Fut, α -1,3-Fut, lacY and SetA in the genetically engineered strain is up-regulated.
7. The method for constructing a genetically engineered strain according to claim 6, comprising transforming or transfecting the recombinant plasmid according to any one of claims 3 to 5 into a host strain.
A method for producing 2 '-fucosyllactose, 3-fucosyllactose and/or difucosyllactose, comprising culturing the genetically engineered strain of claim 6 or the genetically engineered strain obtained by the construction method of claim 7 to obtain a culture containing the 2' -fucosyllactose, 3-fucosyllactose and/or difucosyllactose.
9. The method according to claim 8, wherein glucose and/or lactose are included in the culture medium.
10. The product for improving intestinal immunity is characterized in that the raw materials comprise at least one of the following 1) to 4):
1) The recombinant plasmid according to any one of claims 3 to 5;
2) The genetically engineered strain of claim 6;
3) A genetically engineered strain obtained by the construction method of claim 7
4) A culture produced by the production method according to claim 8 or 9.
CN202311080390.3A 2023-08-25 2023-08-25 Recombinant E.coli for producing complex fucosylated lactose Pending CN117025650A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117467594A (en) * 2023-12-26 2024-01-30 态创生物科技(广州)有限公司 Genetically engineered bacterium for producing 2' -fucosyllactose and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117467594A (en) * 2023-12-26 2024-01-30 态创生物科技(广州)有限公司 Genetically engineered bacterium for producing 2' -fucosyllactose and application thereof
CN117467594B (en) * 2023-12-26 2024-04-09 态创生物科技(广州)有限公司 Genetically engineered bacterium for producing 2' -fucosyllactose and application thereof

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