A fucosyl transferase and 2apos; process for the preparation of fucosyllactose

Abstract

The invention relates to the technical field of biology, in particular to a preparation method of fucosyltransferase and 2 '-fucosyllactose. The invention provides a fucosyltransferase, a nucleic acid molecule, a combined element, a host and a preparation method of 2 '-fucosyllactose. According to the invention, through knockout of a metabolic intermediate degradation gene and overexpression of a 2 '-fucosyllactose biosynthetic pathway, the yield of a 2'-fucosyllactose production engineering strain is greatly improved, and the economical efficiency of synthesis of 2 '-fucosyllactose by a fermentation method is improved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing fucosyltransferase and 2'-fucoyllactose. Background Technology

[0002] 2'-Fucosyllactose (2'-FL) is an oligosaccharide found in human breast milk. It is a disaccharide molecule composed of fucose and lactose. 2'-FL is one of the most abundant oligosaccharides in breast milk, with a content as high as 2-3 g / L.

[0003] 2'-FL plays an important physiological role in early infant growth and development. It has various prebiotic effects, helping to maintain gut health and normal immune function development. Here are some of the main functions and benefits of 2'-FL:

[0004] Prebiotic effects: 2'-FL is a prebiotic that promotes the growth of beneficial bacteria in the gut, such as Bifidobacteria and Rhodopsin. These beneficial bacteria are crucial for the digestion and absorption of nutrients, maintaining intestinal barrier function, and resisting the invasion of harmful bacteria.

[0005] Immune regulation: 2'-FL plays a regulatory role in the development and function of the immune system. It can enhance an infant's natural immunity, improve antiviral and antibacterial capabilities, and reduce the risk of intestinal inflammatory responses.

[0006] Anti-adhesion: 2'-FL can bind to the toxins of some pathogens, preventing them from adhering to intestinal epithelial cells, thereby reducing the risk of infection.

[0007] Nutrient absorption: 2'-FL helps enhance the absorption of nutrients such as calcium and iron, promoting the development of the infant's bones and nervous system.

[0008] Due to the important role of 2'-FL in breast milk, scientists and biotechnology companies are working to develop methods to synthesize this oligosaccharide. Currently, the main synthetic methods include chemical synthesis and yeast-engineered biosynthesis.

[0009] In existing chemical synthesis techniques, most routes utilize fully protected L-fucose donors and lactose acceptors. Differences between methods involve varying protecting group strategies, glycosylation methods, and final preservation strategies. The chemical synthesis of 2'-fucosyllactose was first published in 1981, using a 6-O-benzoylated lactose acceptor and a 3-O-benzylated α-pyranofosyl bromide donor, followed by successive removal of the protecting group. A 2010 patent for the chemical synthesis of 2'-fucosyllactose used a novel intermediate called the O-benzyl / substituted O-benzylated 2'-O-fucosyllactose intermediate. This intermediate possesses crystallization properties that aid in efficient purification methods, allowing for the separation of chemical steps and enabling scale-up production. In 2019, K. Agoston, MJ Hederos, et al., reported a process for producing kilogram-scale 2'-FL. However, chemical methods suffer from drawbacks such as the use of large amounts of organic reagents, complex procedures, harsh reaction conditions, and low product yields. Therefore, the chemical synthesis of 2'-FL using L-fucose as a starting substrate is costly. Furthermore, the lack of low-cost purification methods makes it difficult to achieve stable gram-scale preparations using chemical synthesis.

[0010] The advantages of enzymatic synthesis of oligosaccharides include a certain degree of stereoselectivity and regioselectivity. Two commonly used classes of enzymes in oligosaccharide synthesis include glycosyltransferases (EC 2.4) and glycosidases (EC 3.2.1). Glycosyltransferases (GTs) transfer a monosaccharide molecule from a nucleotide glycosyl donor to an acceptor. The main disadvantages of these enzymes are low stability, difficulty in obtaining them, and the need for expensive nucleotide glycosyl donors. Glycosidases (GHs) are generally used to catalyze the hydrolysis of glycosidic bonds; under specific conditions, they can also catalyze the transglycosylation reaction of activated glycosyl donors to synthesize glycosidic bonds. Because the synthesized products are further hydrolyzed, the yield of transglycosylation products is generally less than 40%–50%. Different fucosyltransferases (FU T) can specifically synthesize glycosidic bonds such as α-1,2, α-1,3, α-1,4, and α-1,6. α-1,2-fucosyltransferase catalyzes the substitution of groups between GDP-fucose and lactose to produce 2'-FL and guanosine diphosphate (GDP). Due to its high cost, the production of 2'-FL using GDP-fucose as a starting material is limited. Some studies have explored the synthesis of 2'-FL using a three-step enzymatic method with lower-cost GDP-D-mannose as a starting substrate. First, the enzyme Gmd (GDP-mannose 4,6-dehydratase, Escherichia coli K-12) catalyzes the synthesis of GDP-4-keto-6-deoxymannose from GDP-D-mannose. Second, the enzyme WcaG (GDP-fucose synthase, Escherichia coli K-12) catalyzes the synthesis of GDP-fucose from GDP-4-keto-6-deoxymannose and NADPH (yield 78%). Third, α-1,2-fucosyltransferase (FucT2, H. pylori) catalyzes the synthesis of 2'-FL from GDP-fucose and lactose (yield 65%). However, this method is relatively complex and difficult to apply. α-L-fucosidase (EC 3.2.1.51) is a class of exoglycoside hydrolases that specifically hydrolyze fucose linked to fucosyl oligosaccharides or other fucosyl compounds. Studies have shown that *Alcaligenes sp.* α-L-fucosidase can catalyze the synthesis of 3'-FL from 4-p-nitrophenyl-α-L-fucoside (pNP-FUC) and lactose with a yield of 34%. *Fusarium gramine arum* (F. graminearum) α-L-fucosidase can synthesize 2'-FL using xylglucan (XyG) and lactose as substrates with a yield of 14%. However, the transglycoside efficiency of these enzymes is low, making them unsuitable for large-scale FL synthesis.Therefore, before discovering α-L-fucosidases with high transglycosylation activity, good stereoselectivity, and regioselectivity, the enzymatic synthesis of 2'-FL also faces the problem of excessive cost.

[0011] The fermentation synthesis of 2'-FL mainly involves the microorganisms themselves or mimicking metabolic mechanisms to synthesize GDP-fucose and then using heterologously expressed α-1,2-fucosyltransferase to catalyze the production of 2'-FL. The functional expression of α-1,2-fucosyltransferase within cells, the accumulation of GDP-fucose within cells, the transferable and accumulative lactose (glycosyl acceptor), and the reduction of the inhibitory effect of byproducts on related enzymes are all key factors in the whole-cell synthesis of 2'-FL. The fermentation synthesis of 2'-FL involves two important steps: (1) the synthesis of the important intermediate substrate GDP-fucose; and (2) the fucosylation of lactose. α-1,2-fucosyltransferase is the key enzyme in the whole-cell synthesis of 2'-FL. Studies have screened the ability of α-1,2-fucosyltransferases from different sources to synthesize 2'-FL, including α-1,2-fucosyltransferases from Helicobacter pylori (H. pylori), Helicobacter mustelae (H. musttelae), Helicobacter bilis (H. bilis), E. coli O128:Bl2, E. coli O86, E. coli O127, C. jejuni, Bacteroides fragilis (B. frag ilis), and Bacteroides vulgatus (B. vulgatus) ATCC8482. The results showed that H. pylori α-1,2-fucosyltransferase (FucT2) had the highest 2'-FL synthesis yield. Currently, most reports and industrial production of 2'-FL use the α-1,2-fucosyltransferase FucT2. However, the extremely low soluble expression of this enzyme in E. coli severely limits its catalytic efficiency, making it the rate-limiting step in 2'-FL production.

[0012] Another key factor in the fermentation production of 2'-FL is increasing the intracellular concentration of GDP-fucose. There are two biosynthetic pathways for GDP-fucose: the de novo pathway and the salvage pathway. Methods to increase GDP-fucose levels in the de novo pathway include: overexpressing genes related to various enzymes that convert mannose-1-phosphate to fructose-6-phosphate and GDP-fucose (manB, manC, gmd, and wcaG); overexpressing the rcsA (or rcsB) gene (a positive regulator of the clonate operon in E. coli); knocking out the wcaJ gene (a gene related to clonate synthesis); and upregulating gene expression related to the ratio between reduced coenzyme II (NADPH) and NADP+. Methods to increase GDP-fucose concentration in the salvage pathway include: overexpressing the L-fucose transporter gene (fucP) in *E. coli* and the fucokinase / fucose-1-phosphate guanylate transferase gene in *B. fragilis*; knocking out enzyme genes involved in L-fucose metabolism, such as fucose-1-phosphate aldolase (fucA), fucose isomerase (fucI), and fucokinase (fucK); and overexpressing genes involved in guanosine triphosphate (GTP) biosynthesis, such as gsk, gpt, gmk, and ndk. Methods to increase intracellular lactose (receptor) concentration include knocking out the intracellular lactase gene (lacZ) and mutating the lactose acetyltransferase gene (lacA) and the lon gene.

[0013] Currently, salvage synthesis of GDP-fucose can achieve higher yields in the fermentation production of 2'-FL. Using L-fucose as a starting substrate has less impact on bacterial metabolism but is more expensive. Overexpression of certain genes in the de novo synthesis pathway can disrupt the host's metabolic balance, affecting bacterial growth and limiting further increases in GDP-fucose and 2'-FL yields.

[0014] Currently, a research team at Seoul National University in South Korea has increased the yield of 2'-FL to 47 g / L in fed-batch fermentation by heterologous expression of FucT2 in E. coli and co-expression of de novo and compensatory pathways. Meanwhile, a team at Kangnam University reported a yield of 100 g / L by expressing a FucT2 homozyme in E. coli. However, challenges remain, including long fermentation times and low production efficiency.

[0015] The production of 2'-fucoyllactose using *E. coli* has become a mature technology. Numerous reports indicate that the final step in 2'-fucoyllactose biosynthesis—the catalytic step of fucosyltransferase—is the rate-limiting step in 2'-fucoyllactose production. This is because the catalytic efficiency of fucT2 is relatively low, requiring further modification or the search for highly efficient homologous enzymes. Furthermore, both fucT2 and its homologous enzymes have poor solubility. Summary of the Invention

[0016] In view of this, the present invention provides a method for preparing fucosyltransferase and 2'-fucoyllactose. The present invention provides fucosyltransferase, nucleic acid molecules, combinatorial elements, a host, and a method for preparing 2'-fucoyllactose. By knocking out genes that degrade metabolic intermediates and overexpressing the 2'-fucoyllactose biosynthetic pathway, the present invention significantly increases the yield of engineered strains producing 2'-fucoyllactose and improves the economic efficiency of fermentation-based synthesis of 2'-fucoyllactose.

[0017] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0018] This invention provides a fucosyltransferase having:

[0019] (I) An amino acid sequence as shown in SEQ ID NO:1; or

[0020] (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0021] (III) Sequences that are more than 90% homologous to the amino acid sequences shown in (I) or (II).

[0022] In some specific embodiments of the present invention, the fucosyltransferase further includes an ADK tag; the amino acid sequence of the ADK tag is shown in SEQ ID NO: 2.

[0023] In some specific embodiments of the present invention, the ADK tag is located at the N-terminus of the nucleic acid molecule.

[0024] In some specific embodiments of the present invention, the fucosylation enzyme has the following characteristics:

[0025] (I) An amino acid sequence as shown in SEQ ID NO:3; or

[0026] (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0027] (III) Sequences that are more than 90% homologous to the amino acid sequences shown in (I) or (II).

[0028] The present invention also provides a complex enzyme comprising the aforementioned fucosyltransferase, phosphogmannanase, mannose-1-phosphoganyltransferase, GDP-mannose 4,6-dehydratase, and GDP-L-fucosyl synthase.

[0029] In some specific embodiments of the present invention, the phosphogmannanase, mannose-1-phospguanylate transferase, GDP-mannose 4,6-dehydratase and GDP-L-fucose synthase sequentially possess the following properties:

[0030] (I) The amino acid sequence shown in SEQ ID NO:4-7; or

[0031] (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or

[0032] (III) Sequences that are more than 90% homologous to the amino acid sequences shown in (I) or (II).

[0033] The present invention also provides a nucleic acid molecule encoding the fucosylation enzyme.

[0034] In some specific embodiments of the present invention, the nucleic acid molecule encoding the fucosylation enzyme has:

[0035] (I) A nucleotide sequence as shown in SEQ ID NO:8; or

[0036] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0037] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0038] (IV) A nucleotide sequence that is more than 90% homologous to the nucleotide sequences described in (I), (II) or (III).

[0039] The present invention also provides a combination element comprising the nucleic acid molecule, a nucleic acid molecule encoding phosphomannanase, a nucleic acid molecule encoding mannose-1-phosguanosyltransferase, a nucleic acid molecule encoding GDP-mannose 4,6-dehydratase, and a nucleic acid molecule encoding GDP-L-fucose synthase.

[0040] In some specific embodiments of the present invention, the nucleic acid molecule encoding phosphomannanase, the nucleic acid molecule encoding mannose-1-phosphoguanosyltransferase, the nucleic acid molecule encoding GDP-mannose 4,6-dehydratase, and the nucleic acid molecule encoding GDP-L-fucose synthase sequentially have:

[0041] (I) Nucleotide sequences as shown in SEQ ID NO:9-12; or

[0042] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0043] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0044] (IV) A nucleotide sequence that is more than 90% homologous to the nucleotide sequences described in (I), (II) or (III).

[0045] The present invention also provides a recombinant vector, including any of the following, and a scaffold vector:

[0046] (A) the nucleic acid molecule; and / or

[0047] (B) The combined elements.

[0048] In some specific embodiments of the present invention, the skeleton carrier includes the pGEX series and / or the pET series.

[0049] In some specific embodiments of the present invention, the skeleton carrier includes one or more of pGEX-5x, pET28a, or pAC.

[0050] In some specific embodiments of the present invention, the recombinant vector includes recombinant plasmid pGEX-ADK-FH4 and recombinant plasmid pAC-BCDG;

[0051] The recombinant plasmid pAC-BCDG and the recombinant plasmid pGEX-ADK-FH4 sequentially possess the following characteristics:

[0052] (I) Nucleotide sequences as shown in SEQ ID NO:13 and SEQ ID NO:14; or

[0053] (II) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (I), but differs from the nucleotide sequence shown in (I) due to the degeneracy of the genetic code; or

[0054] (III) A nucleotide sequence obtained by substituting, deleting, or adding one or more nucleotide sequences to the nucleotide sequence shown in (I) or (II), and which has the same or similar function to the nucleotide sequence shown in (I) or (II); or

[0055] (IV) A nucleotide sequence that is more than 90% homologous to the nucleotide sequences described in (I), (II) or (III).

[0056] The present invention also provides a host, including the recombinant vector.

[0057] In some specific embodiments of the present invention, the host is selected from Escherichia coli, Bacillus subtilis, Pichia pastoris, or Saccharomyces cerevisiae.

[0058] Escherichia coli is preferred;

[0059] Further preferred strains are Escherichia coli BL21 Star, W3110, or K-12MG1655.

[0060] The present invention also provides the use of the host in the preparation of 2'-fucoidanactose.

[0061] The present invention also provides a method for preparing 2'-fucoyl lactose by fermenting the host to obtain the 2'-fucoyl lactose.

[0062] In some specific embodiments of the present invention, the fermentation temperature of the host includes 30°C; or

[0063] The fermentation time includes 82 hours; or

[0064] The dissolved oxygen content of the fermentation includes 30%; or

[0065] The pH value of the fermentation includes 7.0.

[0066] In some specific embodiments of the present invention, the preparation method includes: taking the host monoclonal culture in LB liquid medium, culturing at 37°C and 220 rpm for 14 h with shaking, inoculating into fermentation medium at an inoculum of 5 v / v% for fermentation; fermentation temperature of 30°C, dissolved oxygen maintained at 30%, pH maintained at 7.0 with 25% ammonia water, terminating fermentation after 82 h to obtain the 2'-fucoyl lactose.

[0067] In some specific embodiments of the present invention, the fermentation culture medium comprises: 1 g / L citric acid monohydrate, 2 g / L diammonium hydrogen phosphate, 6.75 g / L potassium dihydrogen phosphate, 0.7 g / L magnesium sulfate heptahydrate, 7.5 g / L yeast extract, 20 g / L glycerol, 10 g / L lactose, and 10 mL / L TES trace element solution.

[0068] This invention includes, but is not limited to, the following beneficial effects:

[0069] 1. A novel and highly efficient fucosyltransferase (FH4) has been discovered. FH4, derived from *Phocaeicolacoprocola*, offers improved catalytic efficiency compared to the commonly used *HpFucT* and is the rate-limiting enzyme in 2'-FL production. Using FH4 can increase the conversion rate of 2'-FL.

[0070] 2. An ADK tag was added to the N-terminus of the FH4 amino acid sequence, which increased the enzyme's solubility. FH4 itself has poor solubility; adding the tag allows it to be expressed in a more soluble form, thereby improving its catalytic activity.

[0071] 3. An engineered strain expressing manB / C / gmd / wcaG and FH4 in tandem was constructed. This strain tandemly expresses enzymes and transferases of the GDP-fucose biosynthesis pathway, synergistically increasing 2'-FL production.

[0072] 4. By knocking out and overexpressing genes, the metabolic pathways of the host bacteria were optimized, side effects were reduced, and the conversion rate of raw materials was increased.

[0073] 5. The superior performance of the engineered strain was verified in shake flasks and fermenters. The yield in shake flasks increased by 1.8 times, and the yield in fermenters reached 43.7 g / L, achieving an advanced yield level.

[0074] 6. By comprehensively utilizing homologous enzyme substitution, soluble tagging, metabolic engineering, and high-density fermentation technologies, the microbial synthesis yield and production efficiency of 2'-FL have been significantly improved.

[0075] 7. The process of this invention is simple and controllable, the raw materials are widely available, and it has good application prospects. Attached Figure Description

[0076] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0077] Figure 1 Schematic diagram of recombinant plasmid pGEX-fucT2-P1;

[0078] Figure 2 Schematic diagram of recombinant plasmid pGEX-GST-FH4;

[0079] Figure 3 Schematic diagram of recombinant plasmid pAC-BCDG;

[0080] Figure 4 Schematic diagram of recombinant plasmid pGEX-ADK-FH4;

[0081] Figure 5SDS-PAGE analysis of the target gene expression level in engineered strain 2FL1;

[0082] Figure 6 SDS-PAGE analysis of the target gene expression level in engineered strain 2FL2;

[0083] Figure 7 SDS-PAGE analysis of the target gene lysis level in engineered strain 2FL1;

[0084] Figure 8 SDS-PAGE analysis of the target gene lysis level in engineered strain 2FL2;

[0085] Figure 9 Analysis of the results of shake-flask fermentation of strain 2'-fucolactose;

[0086] Figure 10 Analysis of fermentation results of engineered strain 2FL2 in a fermenter. Detailed Implementation

[0087] This invention discloses a method for preparing fucosyltransferase and 2'-fucoyllactose. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0088] Improving the solubility of α-1,2-fucosyltransferase through homologous enzyme substitution, tag-based solubilization, cofactor expression, and point mutation is an effective means to improve the production efficiency of 2'-FL. Improving solubility and increasing the soluble expression of the enzyme is one feasible solution to address the rate-limiting step in 2'-fucoyllactose biosynthesis. This invention aims to identify a highly efficient FucT2 homologous enzyme catalyzing the rate-limiting step by screening homologous enzymes, and further increase the solubility of the target protein by adding a tag to achieve the goal of increasing 2'-fucoyllactose production.

[0089] The purpose of this invention is to manufacture a recombinant *E. coli* strain capable of tandemly expressing the following genes: phosphogmannanase (manB), mannose-1-phospguanosyltransferase (manC), GDP-mannose 4,6-dehydratase (gmd), GDP-L-fucose synthase (wcaG), and fucosyltransferase (ADK-FH4). By knocking out genes that degrade metabolic intermediates and overexpressing the 2'-fucosyllactose biosynthetic pathway, the inventors significantly increased the yield of the 2'-fucosyllactose-producing strain, thus improving the economic viability of fermentation-based 2'-fucosyllactose synthesis. This invention includes the following technical solutions:

[0090] A fucosyltransferase with an ADK tag, derived from *Phocaeicola coprocola* (DSM 17136), has the amino acid sequence SEQ ID NO:1. A tandemly expressed gene consisting of phosphomannanase (manB), mannose-1-phosphate guanosyltransferase (manC), GDP-mannose 4,6-dehydratase (gmd), and GDP-L-fucose synthase (wcaG) from *Escherichia coli* K-12MG1655, has the amino acid sequences SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively.

[0091] A second aspect of the invention provides two plasmids each containing the ADK-FH4 gene described above, and plasmids expressing the manB, manC, gmd, and wcaG genes in tandem. The plasmids contain a vector backbone for expressing the ADK-FH4 gene, preferably a pGEX series vector such as pGEX-5x, or a pET series vector such as pET28a, but are not limited thereto.

[0092] A third aspect of the present invention is to provide a microorganism for expressing the aforementioned manB, manC, gmd, wcaG, and ADK-FH4 genes. For example, the ADK-FH4 gene is cloned into the genome of this microorganism.

[0093] The aforementioned microbial hosts can be selected from Escherichia coli, Bacillus subtilis, Pichia pastoris, and Saccharomyces cerevisiae, with Escherichia coli being preferred, and Escherichia coli W3110 or K-12MG1655 being more preferred.

[0094] A fourth aspect of the invention provides the use of microorganisms expressing the above-mentioned manB, manC, gmd, wcaG, and ADK-FH4 genes in the production of 2'-fucoidanactose.

[0095] Specifically, 2'-fucoyl lactose is produced through microbial fermentation.

[0096] The experimental methods used in the examples were performed under standard conditions, referring to the experimental methods described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (4th Edition, 2017, Science Press).

[0097] The materials and reagents used in the examples are all commercially available.

[0098] The fucosyltransferase fragment of *Phocaeicola coprocola* used in this example has its amino acid sequence published in the GenB ank sequence database established by the National Center for Biotechnology Information (NCBI), with accession number WP_007567024.1. The physical DNA was synthesized from commercial biotechnology companies, such as GenScript Biotech Inc., after codon optimization. The DNA sequences of manB, manC, gmd, and wcaG were obtained by PCR amplification using genomic DNA from *Escherichia coli* strain K-12MG1655 as a template.

[0099] Fucosyltransferase amino acid sequence (SEQ ID NO:1):

[0100] GGSGGGGSMRLIKMTGGGLGNQMFIYAFYLKMKKLFPHTKIDLSDMMHYHVHHGYEMNRVFALPHTEFCINRTLKKLMEFLLCKVVYERKQKNGSMEAFEKKYAWPLIYFKGFYQSERFFADIEDDVRKTFCFNMELINIRSREMM KVIDADEHAVSIHIRRGDYLLPKFWANAGCVCQLPYYKNAIAELKKHDSTPSFYVFSDDIEWVKQNLSLSNAHYIDWNQGNDSWQDMMLMSHCRNHIICNSTFSWWGAWLNPRKNKTVIVPSRWFMKEETPYIYPARWIKVPIN*

[0101] The amino acid sequence of the ADK tag (SEQ ID NO:2):

[0102] MRIILLGAPGAGKGTQAQFIMEKYGIPQISTGDMLRAAVKSGSELGKQAKDIMDAGKLVTDELVIALVKERIAQEDCRNGFLLDGFPRTIPQADAMKEAGINVDYVLEFDVPDELIVDRIVGRRVHAPSGRVYHVKFNPPKVEGKDDVTGEELTTRKDDQEETVRKRLVEYHQMTAPLIGYYSKEAEAGNTKYAKVDGTKPVAEVRADLEKILG

[0103] Amino acid sequence of fucosyltransferase with ADK tag (SEQ ID NO:3):

[0104] MRIILLGAPGAGKGTQAQFIMEKYGIPQISTGDMLRAAVKSGSELGKQAKDIMDAGKLVTDELVIALVKERIAQEDCRNGFLLDGFPRTIPQADAMKEAGINVDYVLEFDVPDELIVDRIVGRRVHAPSGRVYHVKFNPPKVEGKDDVTGEELTTRKDDQEETVRKRLVEYHQMTAPLIGYYSKEAEAGNTKYAKVDGTKPVAEVRADLEKILGGGSGGGGSMRLIKMTGGLGNQMFIYAFYLKMKKLFPHTKIDLSDMMHYHVHHGYEMNRVFALPHTEFCINRTLKKLMEFLLCKVVYERKQKNGSMEAFEKKYAWPLIYFKGFYQSERFFADIEDDVRKTFCFNMELINIRSREMMKVIDADEHAVSIHIRRGDYLLPKFWANAGCVCQLPYYKNAIAELKKHDSTPSFYVFSDDIEWVKQNLSLSNAHYIDWNQGNDSWQDMMLMSHCRNHIICNSTFSWWGAWLNPRKNKTVIVPSRWFMKEETPYIYPARWIKVPIN*

[0105] Amino acid sequence of phosphomannanase (ManB) (SEQ ID NO:4):

[0106] MKKLTCFKAYDIRGKLGEELNEDIAWRIGRAYGEFLKPKTIVLGGDVRLTSETLKLALAKGLQDAGVDVLDIGMSGTEEIYFATFHLGVDGGIEVTASHNPMDYNGMKLVREGARPISGDTGLRDVQRLAEANDFPPVDETKRGRYQQINLRDAYVDHLFGYINVKNLTPLKLVINSGNGAAGPVVDAIEARFKALGAPVELIKVHNTPDGNFPNGIPNPLLPECRDDTRNAVIKHGADMGIAFDGDFDRCFLFDEKGQFIEGYYIVGLLAEAFLEKNPGAKIIHDPRLSWNTVDVVTAAGGTPVMSKTGHAFIKERMRKEDAIYGGEMSAHHYFRDFAYCDSGMIPWLLVAELVCLKDKTLGELVRDRMAAFPASGEINSKLAQPVEAINRVEQHFSREALAVDRTDGISMTFADWRFNLRTSNTEPVVRLNVESRGDVPLMEARTRTLLTLLNE*

[0107] Amino acid sequence of mannose-1-phosphate guanylyltransferase (ManC) (SEQ ID NO:5):

[0108] MAQSKLYPVVMAGGSGSRLWPLSRVLYPKQFLCLKGDLTMLQTTICRLNGVEMAQSKLYPVVMAGGSGSRLWPLSRVLYPKQFLCLKGDLTMLQTTICRLNGVECESPVVICNEQHRFIVAEQLRQLNKLTENIILEPAGRNTAPAIALAALAAKRHSPESDPLMLVLAADHVIADEDAFRAAVRNAMPYAEAGKLVTFGIVPDLPETGYGYIRRGEVSAGEQDMVAFEVAQFVEKPNLETAQAYVASGEYYWNSGMFLFRAGRYLEELKKYRPDILDACEKAMSAVDPDLNFIRVDEEAFLACPEESVDYAVMERTADAVVVPMDAGWSDVGSWSSLWEISAHTAEGNVCHGDVINHKTENSYVYAESGLVTTVGVKDLVVVQTKDAVLIADRNAVQDVKKVVEQIKADGRHEHRVHREVYRPWGKYDSIDAGDRYQVKRITVKPGEGLSVQMHHHRAEHWVVVAGTAKVTIDGDIKLLGENESIYIPLGATHCLENPGKIPLDLIEVRSGSYLEEDDVVRFADRYGRV*

[0109] Amino acid sequence of GDP - mannose 4,6 - dehydratase (Gmd) (SEQ ID NO:6):

[0110] MSKVALITGVTGQDGSYLAEFLLEKGYEVHGIKRRASSFNTERVDHIYQDPHTCNPKFHLHYGDLSDTSNLTRILREVQPDEVYNLGAMSHVAVSFESPEYTADVDAMGTLRLLEAIRFLGLEKKTRFYQASTSELYGLVQEIPQKETTPFYPRSPYAVAKLYAYWITVNYRESYGMYACNGILFNHESPRRGETFVTRKITRAIANIAQGLESCLYLGNMDSLRDWGHAKDYVKMQWMMLQQEQPEDFVIATGVQYSVRQFVEMAAAQLGIKLRFEGTGVEEKGIVVSVTGHDAPGVKPGDVIIAVDPRYFRPAEVETLLGDPTKAHEKLGWKPEITLREMVSEMVANDLEAAKKHSLLKSHGYDVAIALES*

[0111] GDP-L-fucose synthase (WcaG) amino acid sequence (SEQ ID NO:7):

[0112] MSKQRVFIAGHRGMVGSAIRRQLEQRGDVELVLRTRDELNLLDSRAVHDFFASERIDQVYLAAAKVGGIVANNTYPADFIYQNMMIESNIIHAAHQNDVNKLLFLGSSCIYPKLAKQPMAESELLQGTLEPTNEPYAIAKIAGIKLCESYNRQYGRDYRSV MPTNLYGPHDNFHPSNSHVIPALLRRFHEATAQNAPDVVVWGSGTPMREFLHVDDMAAASIHVMELAHEVWLENTQPMLSHINVGTGVDCTIRELAQTIAKVVGYKGRVVFDASKPDGTPRKLLDVTRLHQLGWYHEISLEAGLASTYQWFLENQDRFRG*

[0113] The nucleotide sequence encoding the fucosyltransferase (SEQ ID NO:8):

[0114] GGCGGTTCTGGCGGTGGCGGTTCTATGCGTCTGATTAAAATGACCGGCGGTCTGGGTAATCAGATGTTCATCTATGCCTTCTATCTGAAAATGAAAAAGCTGTTCCCGCATACCAAAATTGATCTGAGCGATATGATGCATTATCATGTGCATCATGGTTATGAAATGAATCGTGTGTTCGCACTGCCGCATACCGAGTTCTGTATTAATCGCACCCTGAAAAAACTGATGGAGTTCCTGCTGTGCAAAGTGGTGTATGAACGTAAACAGAAAAATGGTAGTATGGAAGCCTTCGAAAAAAAATATGCCTGGCCGCTGATCTACTTCAAAGGCTTCTATCAGAGCGAACGCTTCTTCGCCGATATTGAAGATGATGTGCGCAAAACCTTCTGCTTCAATATGGAACTGATTAATATCCGTAGTCGTGAAATGATGAAAGTTATTGATGCCGATGAACATGCAGTGAGCATTCATATTCGTCGTGGTGATTATCTGCTGCCGAAATTCTGGGCCAATGCCGGTTGTGTGTGTCAGCTGCCGTATTATAAAAATGCAATTGCCGAACTGAAGAAACATGATAGTACCCCGAGCTTCTATGTGTTCAGCGATGATATTGAATGGGTTAAACAGAATCTGAGCCTGAGTAATGCCCATTATATTGATTGGAATCAGGGCAATGATAGTTGGCAGGATATGATGCTGATGAGTCATTGCCGTAATCATATTATCTGTAATAGCACCTTCAGTTGGTGGGGCGCCTGGCTGAATCCGCGCAAAAATAAAACCGTGATTGTTCCGAGTCGTTGGTTCATGAAAGAAGAAACACCTTATATCTATCCGGCCCGCTGGATTAAAGTGCCGATTAATTAA Nucleotide sequence encoding mannan phosphatase (ManB) (SEQ ID NO:9):

[0115]

[0116] The nucleotide sequence encoding mannose-1-phosphate guanosine transferase (ManC) (SEQ ID NO:10):

[0117]

[0118] The amino acid sequence encoding GDP-mannose 4,6-dehydratase (Gmd) (SEQ ID NO:11):

[0119]

[0120] The amino acid sequence encoding GDP-L-fucose synthase (WcaG) (SEQ ID NO:12):

[0121] atgagtaaacaacgagtttttattgctggtcatcgcgggatggtcggttctgccatcaggcggcagctcgaacagcgcggtgatgtggaactggtattacgcacccgcgacgagctgaacctgttggacagccgcgcggtgcatgatttctttgccagcgaacgcattgaccaggtctatctggcggcggcgaaagtgggcggcattgttgctaacaacacctatccggcggatttcatctaccagaacatgatgattgagagcaacatcattcacgccgcgcatcagaacgacgtgaacaaactgctgtttctcggatcgtcctgtatctacccgaaactggcaaaacagccgatggcagaaagcgagttgttgcagggcacgctggagccgactaacgagccttatgctattgccaaaatcgccgggatcaaactgtgcgaatcttacaatcgccagtacggacgagattaccgttcagtcatgccgaccaacctgtacgggccgcacgacaacttccacccgagtaattcgcatgtgatcccagcattgctgcgccgcttccacgaggcgacggcacagaatgcaccggacgtggtggtatggggcagcggtacaccgatgcgtgaattcctgcacgtcgatgatatggcggcggcgagcattcatgtcatggagctggcgcatgaagtctggctggagaacacccagccgatgctgtcgcacattaacgtcggcacgggcgttgactgcaccatccgtgaactggcgcaaaccatcgccaaagtggtgggttacaaaggtcgggtggtttttgatgccagcaaaccggatggtacgccgcgcaaactgctggatgtgacgcgcctgcatcagcttggctggtatcacgaaatctcactggaagcggggcttgccagcacttaccagtggttccttgagaatcaagaccgctttcgggggtaa

[0122] Recombinant plasmid pAC-BCDG nucleotide sequence (SEQ ID NO:13):

[0123]

[0124] The sequence of recombinant plasmid pGEX-ADK-FH4 (SEQ ID NO:14):

[0125] AGCTTATCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGTATTCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGATGCGTATCATTCTGCTTGGCGCTCCGGGCGCGGGGAAAGGGACTCAGGCTCAGTTCATCATGGAGAAATATGGTATTCCGCAAATCTCCACTGGCGATATGCTGCGTGCTGCGGTCAAATCTGGCTCCGAGCTGGGTAAACAAGCAAAAGACATTATGGATGCTGGCAAACTGGTCACCGACGAACTGGTGATCGCGCTGGTTAAAGAGCGCATTGCTCAGGAAGACTGCCGTAATGGTTTCCTGTTGGACGGCTTCCCGCGTACCATTCCGCAGGCAGACGCGATGAAAGAAGCGGGCATCAATGTTGATTACGTTCTGGAATTCGACGTACCGGACGAACTGATTGTTGATCGTATCGTAGGCCGCCGCGTTCATGCGCCGTCTGGTCGTGTTTATCACGTTAAATTCAATCCGCCGAAAGTAGAAGGCAAAGACGACGTTACCGGTGAAGAACTGACTACCCGTAAAGACGATCAGGAAGAAACCGTGCGTAAACGTCTGGTTGAATACCATCAGATGACTGCACCGCTGATCGGCTACTACTCCAAAGAAGCGGAAGCGGGTAACACCAAATACGCGAAAGTTGACGGCACCAAGCCGGTTGCTGAAGTTCGCGCTGATCTGGAAAAAATCCTCGGC GGCGGTTCTGGCGGTGGCGGTTCTATGCGT CTGATTAAAATGACCGGCGGTCTGGGTAATCAGATGTTCATCTATGCCTTCTATCTGAAAATGAAAAAG CTGTTCC CGCATACCAAAATTGATCTGAGCGATATGATGCATTATCATGTGCATCATGGTTATGAAATGAATCGTGTGTTCGC ACTGCCGCATACCGAGTTCTGTATTAATCGCACCCTGAAAAAACTGATGGAGTTCCTGCTGTGCAAAGTGGTGTAT GAACGTAAACAGAAAAATGGTAGTATGGAAGCCTTCGAAAAAAAATATGCCTGGCCGCTGATCTACTTCAAAGGCT TCTATCAGAGCGAACGCTTCTTCGCCGATATTGAAGATGATGTGCGCAAAACCTTCTGCTTCAATATGGAACTGAT TAATATCCGTAGTCGTGAAATGATGAAAGTTATTGATGCCGATGAACATGCAGTGAGCATTCATATTCGTCGTGGT GATTATCTGCTGCCGAAATTCTGGGCCAATGCCGGTTGTGTGTGTCAGCTGCCGTATTATAAAAATGCAATTGCCG AACTGAAGAAACATGATAGTACCCCGAGCTTCTATGTGTTCAGCGATGATATTGAATGGGTTAAACAGAATCTGAG CCTGAGTAATGCCCATTATATTGATTGGAATCAGGGCAATGATAGTTGGCAGGATATGATGCTGATGAGTCATTGC CGTAATCATATTATCTGTAATAGCACCTTCAGTTGGTGGGGCGCCTGGCTGAATCCGCGCAAAAATAAAACCGTGA TTGTTCCGAGTCGTTGGTTCATGAAAGAAGAAACACCTTATATCTATCCGGCCCGCTGGATTAAAGTGCCGATTAA TTAA

[0126] plasmid pGEX-FH4 sequence (SEQ ID NO:15):

[0127]

[0128] The nucleotide sequence (SEQ ID NO:16) encoding the fucosyltransferase (HpFucT):

[0129] ATGGCCTTTAAAGTTGTGCAGATTTGTGGCGGTCTGGGTAATCAGATGTTTCAGTATGCATTTGCAAAAAGCCTGCAGAAACATCTGAATACCCCGGTGCTGCTGGATACCACCAGTTTTGATTGGAGCAATCGTAAAATGCAGCTGGAACTGTTTCCGATTGATCTGCCGTATGCCAATGCCAAAGAAATTGCCATTGCAAAAATGCAGCATCTGCCGAAACTGGTGCGTGATGCACTGAAATATATTGGCTTTGATCGTGTGAGCCAGGAAATTGTTTTTGAATATGAACCGAAACTGCTGAAACCGAGTCGCCTGACCTATTTCTTTGGTTATTTTCAGGATCCGCGTTATTTTGATGCCATTAGTAGCCTGATTAAGCAGACCTTTACCCTGCCGCCGCCGCCGGAAAATAATAAGAATAATAATAAGAAGGAGGAGGAGTATCAGCGCAAACTGAGCCTGATTCTGGCAGCAAAAAATAGCGTTTTTGTGCATATTCGTCGTGGTGACTATGTTGGCATTGGCTGCCAGCTGGGTATTGATTATCAGAAAAAAGCCCTGGAATATATGGCAAAACGTGTTCCGAATATGGAACTGTTTGTTTTCTGTGAAGATCTGAAATTCACTCAGAATCTGGATCTGGGTTATCCGTTTACCGATATGACCACCCGCGATAAAGAAGAAGAAGCATATTGGGATATGCTGCTGATGCAGAGCTGTAAACATGGCATTATTGCAAATAGCACCTATAGCTGGTGGGCAGCCTATCTGATGGAAAATCCGGAAAAAATTATCATTGGCCCGAAACATTGGCTGTTTGGCCATGAAAATATTCTGTGTAAAGAATGGGTGAAAATCGAAAGTCATTTTGAAGTGAAAAGCCAGAAATATAACGCATAA

[0130] Unless otherwise specified, the raw materials and reagents used in the preparation methods of fucosyltransferase and 2'-fucoyllactose provided by this invention can all be purchased from the market.

[0131] The present invention will be further illustrated below with reference to the embodiments:

[0132] Example 1 Construction and Identification of Recombinant Strains

[0133] 1. Construction of recombinant plasmids containing pGEX-fucT2-P1, pGEX-GST-FH4, and pAC-BCDG

[0134] 1) Construction of recombinant plasmids

[0135] The plasmid pGEX-5x is 5.8 kb in size and contains the ampicillin resistance gene, the lactose repressor lac I gene, the ori replication origin, the tac promoter, and multiple restriction endonuclease sites. The plasmid pAC is 2.4 kb in size and contains the chloramphenicol resistance gene, the p15A ori replication origin, the tac promoter, and multiple restriction endonuclease sites.

[0136] The target DNA fragment was ligated into a vector using a homologous recombination kit (Beyotime Biotechnology, catalog number BL1046B-1). The reaction mixture consisted of 10 μl of gene cluster DNA, 1 μl of plasmid DNA, 1 μl of ligase, 2 μl of buffer, and 6 μl of sterile water. The mixture was incubated at 50°C for 30 minutes. The ligation product was transformed into *E. coli* host bacteria DH5α (Thermo Fisher Scientific, catalog number 18265017) using the heat shock method. The transformed product was then plated onto LB agar containing 1% peptone, 0.5% yeast extract, 1% sodium chloride, and 1.5% agar powder and kanamycin.

[0137] Specifically, a commercially synthesized fucosyltransferase gene DNA fragment (nucleotide sequence as shown in SEQ ID NO:8) was ligated into the pGEX-5x plasmid to obtain the recombinant plasmid pGEX-GST-FH4 (SEQ ID NO:15); a commercially synthesized fucosyltransferase gene DNA fragment (HpFucT nucleotide sequence) was ligated into the pGEX-5x plasmid to obtain the recombinant plasmid pGEX-fucT2-P1; and DNA fragments of manB (nucleotide sequence as shown in SEQ ID NO:9), manC (nucleotide sequence as shown in SEQ ID NO:10), gmd (nucleotide sequence as shown in SEQ ID NO:11), and wcaG (nucleotide sequence as shown in SEQ ID NO:12) were ligated into the pAC plasmid to obtain the recombinant plasmid pAC-BCDG (nucleotide sequence as shown in SEQ ID NO:8). As shown in NO:12); schematic diagrams of recombinant plasmids pGEX-fucT2-P1, pGEX-GST-FH4 (pGEX-FH4), and pAC-BCDG (pAC-manCB-Gmd-WcaG) are shown respectively. Figure 1 , Figure 2 , Figure 3 As shown.

[0138] 2) Identification of recombinant plasmids

[0139] The LB plates containing the spread LB solid medium were incubated at 37°C until transformants were grown. Single colonies were picked and incubated overnight at 37°C in LB liquid medium. After centrifugation at 12,000 rpm for 1 minute, plasmids were extracted using a rapid plasmid mini-extraction kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number DP105-03). The extraction method was performed according to the kit instructions.

[0140] The recombinant vector was digested with EcoRI and NdeI. The digestion system was: DNA 43 μL, buffer R 5 μL, NdeI 1 μL, EcoRI 1 μL, and incubated at 37°C for 3 hours. Electrophoresis was used to identify the DNA fragment containing the target 2'-fucosylation-lactose synthesis gene cluster. The target plasmid sequence was verified by sequencing.

[0141] 2. Construct production strains containing recombinant vectors pGEX-fucT2-P1, pAC-BCDG, pGEX-GST-FH4, and pAC-BCDG.

[0142] The recombinant plasmids pGEX-GST-FH4, pAC-BCDG, pGEX-fucT2-P1, and pAC-BCDG obtained in step 1 were transformed into *E. coli* host strain BL21 Star (purchased from Thermo Fisher Scientific, catalog number C601003) using the heat shock method. The transformed bacteria were plated onto LB agar plates containing 1% peptone, 0.5% yeast extract, 1% sodium chloride, and 1.5% agar powder. The LB plates containing the plated agar were incubated at 37°C until transformants appeared. Single colonies were picked to obtain the expected engineered control strain 2FLA and the engineered strain 2FL1.

[0143] Example 2: Tag-optimized FH4 gene expression recombinant plasmid construction and identification.

[0144] 1. Construction of recombinant plasmid pGEX-ADK-FH4

[0145] The original GST tag was replaced with an ADK tag to obtain the recombinant plasmid pGEX-ADK-FH4. The nucleotide sequence of the recombinant plasmid pGEX-ADK-FH4 is shown in SEQ ID NO:14 of the sequence listing. A schematic diagram of the recombinant plasmid pGEX-ADK-FH4 is shown below. Figure 4 As shown.

[0146] 2. Construct a production strain containing the recombinant vectors pGEX-ADK-FH4 and pAC-BCDG.

[0147] The pGEX-ADK-FH4 plasmid and the recombinant plasmid pAC-BCDG described in Example 1 were transformed into the Escherichia coli host strain BL21 Star (purchased from Thermo Fisher Scientific, catalog number C601003) using the heat shock method. The transformed bacteria were plated onto LB agar plates containing 1% peptone, 0.5% yeast extract, 1% sodium chloride, and 1.5% agar powder. The LB plates were incubated at 37°C until transformants appeared. Single colonies were picked to obtain the desired engineered strain 2FL2.

[0148] Example 3: Validation of expression levels in production strains containing the pGEX-ADK-FH4 and pAC-BCDG recombinant vectors

[0149] Detection of target gene expression levels

[0150] Ten single clones each of engineered bacteria 2FL1 constructed in Example 1 and engineered bacteria 2FL2 constructed in Example 2 were selected and cultured in 5 mL LB medium at 37°C and 220 rpm until the OD600 reached approximately 0.6–0.8. IPTG was added to a final concentration of 0.3 mM, and the culture was continued at 37°C and 220 rpm for 18 h. The cells were centrifuged at 4°C and 5000 rpm for 5 min, and the supernatant was discarded. The bacterial cells were resuspended in 2 mL PBS (20 mM, pH 7.0) buffer and then sonicated (40 rpm power, 3 s disruption time, 2 s interval, total 10 min). SDS-PAGE analysis was performed. All target genes were expressed: see [link to relevant documentation]. Figures 5-6 .

[0151] Example 4: Verification of solubility levels in production strains containing pGEX-ADK-FH4 and pAC-BCDG recombinant vectors

[0152] Two single clones were selected from engineered bacteria 2FL1 constructed in Example 1 and engineered bacteria 2FL2 constructed in Example 2 and added to 5 mL of LB medium. They were cultured at 37 °C and 220 rpm until the OD600 reached approximately 0.6–0.8. IPTG inducer was added to a final concentration of 0.3 mM, and the culture was continued at 37 °C and 220 rpm for 18 h. The cells were centrifuged at 4 °C and 5000 rpm for 5 min, and the supernatant was discarded, retaining the bacterial cells. The bacterial cells were resuspended in 2 mL of PBS (20 mM, pH 7.0) buffer and then sonicated (40 rpm power, 3 s disruption time, 2 s interval, total 10 min) to obtain whole cell lysate. The whole cell lysate was centrifuged at 12000 rpm for 3 min to obtain the lysate supernatant and lysate precipitate. The lysate supernatant (S) and lysate precipitate (I) were analyzed by SDS-PAGE.

[0153] Comparing the soluble expression percentages of recombinant proteins from different strains. Specifically, the size of the "S" band and the "I" band in the SDS-PAGE protein gel were compared. The soluble percentage of ADK-FH4 in engineered strain 2FL2 was significantly higher than that of GST-FH4 in engineered strain 2FL1 (the "S" band in the supernatant was larger): see [link to previous section]. Figures 7-8 The “I” band represents the SDS-PAGE analysis result of the precipitate of the broken liquid, and the “S” band represents the SDS-PAGE analysis result of the supernatant of the broken liquid.

[0154] Example 5: Shake-flask fermentation of 2'-fucolactose engineered strains

[0155] The engineered control strain 2FLA and engineered strain 2FL1 constructed in Example 1, and the engineered strain 2FL2 constructed in Example 2, were streaked on LB agar plates and incubated at 37°C for approximately 16 hours. Three single colonies were then transferred to LB tubes and cultured at 37°C with shaking at 220 rpm for approximately 14 hours. The inoculum was then transferred to shake flasks at a 1 v / v ratio and cultured at 37°C with shaking at 220 rpm for approximately 12 hours. Finally, a 4 v / v inoculum was transferred to fermentation shake flasks and cultured at 30°C with shaking at 220 rpm for approximately 72 hours. The shake flask culture medium consisted of: 1 g / L citric acid monohydrate, 2 g / L diammonium hydrogen phosphate, 6.75 g / L potassium dihydrogen phosphate, 0.7 g / L magnesium sulfate heptahydrate, 7.5 g / L yeast extract, 20 g / L glycerol, 10 g / L lactose, 10 mL / L TES trace element solution, and water to a final volume of 1 L. Samples were taken to determine the content of 2'-fuco-lactose. The fermentation results are shown in [Figure 1]. Figure 9 Table 1.

[0156] Table 1. Experimental results of 2'-fucolactose production by shake-flask fermentation using strain 1.

[0157] strain name Group 1 Group 2 Group 3 Average yield (g / L) Conversion rate (lactose molar ratio) 2FLA 0.38 0.32 0.30 0.33 0.47 2FL1 4.45 4.15 4.30 4.30 0.57 2FL2 7.76 7.26 8.26 7.76 0.69

[0158] Depend on Figure 9 As shown in Table 1, the genetically engineered strain 2FL2 constructed in this invention improved the expression level and solubility of the target gene in the starting strain 2FL1, thereby enabling more efficient conversion of lactose into 2'-fucolactose. The average yield of the engineered strain 2FL2 in shake flasks was 1.8 times that of 2FL1. Calculations showed a highly significant difference in shake flask fermentation yield between the two engineered strains (P < 0.01), indicating that the yield of 2-FL2 was significantly higher than that of 2FL1. The enhanced soluble expression of the FH4 gene is beneficial for improving the biosynthesis of 2'-fucolactose.

[0159] Example 6: Fermentation yield verification of 2'-fucolactose engineered strain

[0160] Single clones of the engineered strain 2FL2 constructed in Example 2 and the control homologous enzyme strain FH2 2FLB were picked from LB plates and transferred to LB shake flasks. The flasks were incubated at 37°C with shaking at 220 rpm for approximately 14 hours. A 5 v / v% inoculum was then transferred to a 5 L fermenter. The culture medium composition was as follows: 1 g / L citric acid monohydrate, 2 g / L diammonium hydrogen phosphate, 6.75 g / L potassium dihydrogen phosphate, 0.7 g / L magnesium sulfate heptahydrate, 7.5 g / L yeast extract, 20 g / L glycerol, 10 g / L lactose, and 10 mL / L TES trace element solution. Fermentation was maintained at 30°C, dissolved oxygen at 30%, and pH 7.0 with 25% ammonia. Fermentation was terminated after 82 hours. The 2'-fuco-lactose content of the fermentation broth was periodically detected by HPLC. The fermentation results are shown in [Figure number missing]. Figure 10In this study, the 2'-fucolactose yield of the 2FL2 engineered strain was 43.7 g / L, which is 3.3 times that of the 13.2 g / L of the 2'-fucolactose yield of the 2FLB engineered strain. Compared with the control group homologous enzyme engineered strain 2FLB, it has a certain yield advantage.

[0161] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Fucosyltransferase, characterized in that, It has the following characteristics: (I) An amino acid sequence as shown in SEQ ID NO:1; or (II) A sequence based on the amino acid sequence shown in (I) by substitution, deletion, addition, and / or replacement of one or more amino acids; or (III) Sequences that are more than 90% homologous to the amino acid sequences shown in (I) or (II).

2. The fucosyltransferase according to claim 1, characterized in that, It also includes an ADK tag; the amino acid sequence of the ADK tag is shown in SEQ ID NO:

2.

3. A complex enzyme, characterized in that, Includes the fucosyltransferase, phosphogmannanase, mannose-1-phosphoganyltransferase, GDP-mannose 4,6-dehydratase and GDP-L-fucosyl synthase as described in claim 1 or 2.

4. A nucleic acid molecule encoding the fucosyltransferase as described in claim 1 or 2.

5. A combined element, characterized in that, This includes the nucleic acid molecules as described in claim 4, nucleic acid molecules encoding phosphomannanase, nucleic acid molecules encoding mannose-1-phosguanosyltransferase, nucleic acid molecules encoding GDP-mannose 4,6-dehydratase, and nucleic acid molecules encoding GDP-L-fucose synthase.

6. A recombinant vector, characterized in that, Includes any of the following items and the skeleton carrier: (A) The nucleic acid molecule as described in claim 4; and / or (B) The combined element as described in claim 5.

7. The recombinant vector as described in claim 6, characterized in that, The skeletal carriers include the pGEX series and / or the pET series.

8. The host, characterized in that, Includes the recombinant vector as described in claim 6 or 7.

9. The use of the host as described in claim 8 in the preparation of 2'-fucoidanulose. A method for preparing 10,2'-fucoyl lactose, characterized in that, Fermentation is performed on the host as described in claim 8 to obtain the 2'-fucoyl lactose.

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