Recombinant escherichia coli for producing 2'-fucosyllactose, methods and uses

Abstract

This invention relates to the field of human milk oligosaccharide preparation technology, specifically to a recombinant *Escherichia coli* strain, method, and application for producing 2'-fucosylated lactose. The genome integration and overexpression of the recombinant *E. coli* strain originates from *Azotrophus* spp. (…). Azospirillum sp.) ASfutC Gene, ASfutC The nucleic acid sequence of the gene is shown in SED ID NO:2 or SED ID NO:3. This recombinant *E. coli* strain can efficiently synthesize 2'-fucosylated lactose during fermentation, with a yield of 7.9 g / L, significantly higher than the control strain constructed using other α-1,2-fucosylated transferases. Simultaneously, it generates almost no byproduct DFL, significantly improving the purity of the target product, effectively simplifying subsequent separation and purification processes, reducing production costs, and improving overall production efficiency. It has promising application prospects and significant industrialization value.

Description

Technical Field

[0001] This invention relates to the field of human milk oligosaccharide preparation technology, specifically to a recombinant Escherichia coli for producing 2'-fucosylated lactose, its method, and its application. Background Technology

[0002] Human milk oligosaccharides (HMOs) are the third largest solid component in breast milk after lactose and lipids, playing a vital physiological role in infant growth and development. Numerous studies have shown that HMOs can selectively regulate the gut microbiota of infants, promote the colonization of beneficial bacteria, and have positive effects on brain and nervous system development, immune system maturation and repair. Among the various HMO components, 2'-fucosyllactose (2'-FL) is the most abundant, accounting for approximately 50% of the total fucoidylated oligosaccharides in breast milk. Due to its significant physiological functions and good safety profile, 2'-FL's application value in infant formula and functional foods is increasingly prominent, and international market demand continues to grow.

[0003] Currently, the main methods for producing 2'-FL include chemical synthesis and microbial fermentation. Chemical synthesis is relatively mature, but it typically involves multiple reaction steps and requires the introduction of various protecting groups. This not only results in complex reaction steps and high raw material and operating costs, but also cumbersome subsequent purification processes. Furthermore, the organic solvents and byproducts produced place a significant burden on the environment, making it difficult to meet the demands of large-scale, green production. In contrast, microbial fermentation offers advantages such as mild reaction conditions, a wide range of raw material sources, and environmental friendliness, and is considered a more promising technological route for the industrial production of 2'-FL. However, existing microbial fermentation methods still have significant shortcomings. The α-1,2-fucosyltransferases disclosed for the synthesis of 2'-FL are mainly derived from Helicobacter pylori, such as futC (WP_121037174.1). Although the aforementioned enzymes can catalyze the transfer of GDP-L-fucose to lactose to generate 2'-FL, their overall catalytic activity is low and their substrate specificity is insufficient. During the synthesis of 2'-FL, fucosylation of 2'-FL is easily carried out, generating the byproduct difucosyllactose (DFL). The formation of DFL not only increases the difficulty and cost of subsequent separation and purification processes but also reduces the conversion efficiency of lactose to 2'-FL, thus hindering the efficient industrial production of 2'-FL. Therefore, screening or developing an α-1,2-fucosyltransferase with high catalytic activity and high substrate specificity to achieve efficient and targeted synthesis of 2'-FL, reduce byproduct formation, improve product purity, and lower production costs has become an urgent technical problem to be solved in this field. Summary of the Invention

[0004] To address the technical problems in existing technologies where α-1,2-fucosyltransferase exhibits low catalytic efficiency and poor substrate selectivity during the catalytic synthesis of 2'-FL, and is prone to further fucosylation to generate byproducts such as DFL, resulting in decreased product purity, high subsequent separation and purification costs, and limited industrial conversion efficiency, this invention provides a recombinant Escherichia coli for the production of 2'-fucosyl lactose, along with its method and applications.

[0005] The technical solution of this invention is as follows: In a first aspect, the present invention provides a recombinant *Escherichia coli* strain for producing 2'-fucosylated lactose, wherein the genome is integrated and overexpressed. ASfutC Gene, ASfutC The nucleic acid sequence of the gene is shown as SED ID NO:2 or SED ID NO:3. Preferably, ASfutC The nucleic acid sequence of the gene is shown in SED ID NO:3. By... ASfutC The gene is stably introduced into the host Escherichia coli through genome integration and achieves high-level expression, enabling the obtained recombinant strain to continuously and stably produce ASfutC protein with α-1,2-fucosyltransferase activity, thereby specifically catalyzing the synthesis of 2'-fucosyllactose, effectively improving the synthesis efficiency of the target product and avoiding expression instability caused by plasmid loss.

[0006] Furthermore, recombinant E. coli was used to knock out the β-galactosidase gene. lacZ and UDP-glucose lipid transporter transferase gene wcaJ The *Escherichia coli* MG1655 strain was used as the chassis strain. This chassis strain can improve the utilization efficiency of lactose as an acceptor substrate in the 2'-fucosylation reaction, block the diversion of fucose to the extracellular polysaccharide synthesis pathway, reduce the competitive consumption of fucose substrate, and improve the synthesis flux of 2'-fucosylated lactose from the overall metabolic level.

[0007] Furthermore, ASfutC The integration site of the gene is the *E. coli* MG1655 dadX site. dadX is a non-essential metabolic gene site on the *E. coli* chromosome; its deletion or substitution does not affect the growth and metabolic homeostasis of the host bacteria under nutrient-rich fermentation conditions. The foreign gene... ASfutC Integration into the dadX site enables stable single-copy expression of exogenous genes on chromosomes, avoiding plasmid loss and copy number fluctuations. Furthermore, it eliminates the need for antibiotic selection, thereby improving the genetic stability and industrial applicability of the fermentation process.

[0008] Furthermore, recombinant E. coli utilizes the tac promoter to... ASfutCOverexpression of the gene enables the exogenous gene to achieve a high and controllable transcription level in the host, thereby significantly increasing the expression level of ASfutC protein and increasing the synthesis rate and yield of 2'-fucosylated lactose.

[0009] Furthermore, integration using the CRISPR / Cas9 genome editing system can significantly improve gene editing efficiency and integration accuracy, reduce the occurrence of non-specific mutations, and thus obtain recombinant E. coli strains with clear genetic backgrounds and stable expression.

[0010] Secondly, the present invention provides an application of the above-mentioned recombinant Escherichia coli in the preparation of 2'-fucosylated lactose.

[0011] Thirdly, the present invention provides a method for producing 2'-fucosylated lactose, wherein the above-mentioned recombinant Escherichia coli is used as a fermentation strain to produce 2'-fucosylated lactose in a fermentation system with glycerol and lactose as substrates.

[0012] Furthermore, the fermentation system comprises the following components in the following proportions: glycerol 4 g / L, tryptone 12 g / L, yeast extract 24 g / L, dipotassium hydrogen phosphate 12.54 g / L, potassium dihydrogen phosphate 2.31 g / L, and lactose 20 g / L. This fermentation system provides recombinant *E. coli* with a sufficient and balanced carbon, nitrogen, and inorganic salt environment, which is conducive to maintaining high-density cell growth and efficient expression of ASfutC protein, thereby further improving the yield and purity of 2'-fucosylated lactose and enhancing the stability and reproducibility of the fermentation process.

[0013] The beneficial effects of this invention are as follows: This invention obtained a strain derived from the genus *Azospirobacter* through screening. Azospirillum A novel α-1,2-fucosyltransferase, ASfutC, was developed. This enzyme exhibits significant catalytic activity and strict substrate specificity, specifically catalyzing the conversion of lactose to 2'-fucosyllactose, thus effectively avoiding the formation of the byproduct difucosyllactose (DFL). Codon optimization of the ASfutC protein encoding gene was performed to improve its transcription and translation efficiency in the host microorganism. A recombinant *E. coli* strain capable of efficiently expressing the ASfutC protein was constructed using the optimized encoding gene sequence. This recombinant strain efficiently synthesized 2'-fucosyllactose during fermentation, achieving a yield of 7.9 g / L, significantly higher than the control strain constructed using other α-1,2-fucosyltransferases. Simultaneously, it generated almost no DFL byproduct, significantly improving the purity of the target product, effectively simplifying subsequent separation and purification processes, reducing production costs, and improving overall production efficiency. This recombinant *E. coli* strain shows promising application prospects and significant industrialization value in the industrial production of 2'-fucosyllactose. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 It belongs to the genus *Azospira*. Azospirillum Sequence alignment diagram of ASfutC of α-1,2-glycosyltransferase from *Sp.* and sequence of α-1,2-glycosyltransferase from *Helicobacter pylori* encoding the same enzyme.

[0016] Figure 2 This is a high-performance liquid chromatogram of the remaining amounts of 2'-FL, DFL, and lactose in the fermentation broth of the recombinant strain.

[0017] Figure 3 This is a graph showing the detection results of 2'-FL and DFL content in the fermentation broth of the recombinant strain.

[0018] Figure 4 This is a graph showing the test results of the recombinant strain in a 5L fermenter. Detailed Implementation

[0019] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0020] Example 1: Screening for novel enzymes with α-1,2-fucosyltransferase function In this embodiment, the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) was used. The classic futC (WP_121037174.1) sequence from the glycosyltransferase family was used as the original sequence. The BLAST module on the NCBI website was used for sequential screening, and alignment was performed based on key conserved sites and similarity. The results are as follows: Figure 1 As shown. Thus, a species of *Azospirobacter* was obtained (…). Azospirillum The novel enzyme derived from sp. (NCBI: WP_109047124.1) has the following amino acid sequence: MIIVRLSDGLGNQMFQYAFGRALSTRRGVPLRLDVSAYRVERKRRYELHHFLTEETFVTDEEAHRVITRPHSPDEPWWSQPVVREPHFHYSPDVVQVSSAGYFAGYWQSERHFDDVAPLIRLEFTPKQPLTGANLEVARAIAA RNAVSLHVRRGDYICDPKVNILHGVCSLEYYRAAVAYVAARVEKPEFFVFTDDPDWTRTKLKLDFPAYLVTQNQDAPVEDLRLMTLCRHHIIANSSFSWWGAWLGEKPGQIVCAPQRWFGAYPHDTRDLVPDRWTRLDG (SEQ ID NO:1).

[0021] The novel enzyme derived from *Azospirobacter* was named ASfutC, and its nucleic acid sequence is as follows: ATGATCATCGTCCGCCTGTCCGACGGGCTCGGGAACCAAATGTTCCAGTACGCGTTTGGCCGCGCTCTGTCGACACGGCGCGGCGTGCCCTTGCGCCTAGATGTGTCAGCCTACCGGGTCGAGCGCAAGCGCCGCTACGAGCTTCATCATTTCCTGACCGAAGAAACCTTCGTCACCGATGAAGAGGCGCACCGCGTCATCACCCGACCGCACAGCCCCGACGAACCCTGGTGGAGTCAACCGGTTGTCCGGGAACCGCATTTCCATTACAGCCCCGACGTCGTCCAAGTGTCATCGGCCGGCTATTTCGCCGGCTATTGGCAATCGGAACGACATTTCGACGACGTGGCGCCGCTGATCCGGCTGGAGTTCACCCCGAAGCAGCCGCTGACTGGCGCCAATCTGGAGGTTGCACGGGCGATTGCCGCCCGCAACGCAGTCAGCCTGCATGTGCGCCGCGGTGACTACATCTGCGACCCGAAAGTCAACATCCTGCACGGGGTGTGTTCGCTTGAATATTACCGGGCTGCGGTAGCCTATGTCGCGGCACGGGTGGAAAAACCGGAGTTTTTCGTGTTCACCGACGACCCTGACTGGACACGTACCAAGCTGAAGCTGGATTTCCCAGCCTATCTCGTCACACAGAACCAGGATGCTCCGGTAGAGGATCTTCGTTTGATGACACTGTGCCGTCATCACATCATCGCCAACAGCAGCTTCAGTTGGTGGGGTGCTTGGCTGGGCGAAAAGCCGGGACAGATTGTCTGTGCCCCGCAACGCTGGTTCGGCGCCTATCCGCATGACACCCGCGACTTGGTACCAGACCGCTGGACCCGGCTGGACGGCTGA (SEQ ID NO:2).

[0022] Example 2: Construction of expression vector (1)Codon optimization was performed on the nucleic acid sequence encoding the ASfutC protein (for the specific method, see Patent CN120266212A. The optimized ASfutC nucleic acid sequence is as follows: ATGATTATAGTAAGGCTATCAGATGGATTAGGCAATCAGATGTTCCAGTACGCGTTTGGTCGTGCGCTGTCAACCAGACGTGGTGTTCCGTTGCGTTTAGATGTTTCGGCGTACCGCGTCGAGCGTAAACGTCGCTATGAGCTGCATCACTTCCTGACCGAGGAGACCTTTGTTACCGACGAGGAAGCGCATAGAGTCATCACTCGCCCGCATAGCCCGGACGAGCCGTGGTGGAGTCAGCCGGTTGTGCGTGAACCGCACTTCCATTATAGCCCAGACGTTGTCCAGGTCAGTAGCGCTGGCTACTTCGCCGGTTATTGGCAGAGTGAACGTCACTTCGACGACGTTGCGCCACTCATTCGTCTTGAGTTCACCCCGAAGCAACCGTTGACTGGCGCCAACTTGGAGGTGGCACGCGCCATCGCCGCTCGCAACGCGGTTTCCCTGCATGTTCGTCGCGGTGATTATATTTGCGATCCGAAGGTGAACATTCTGCACGGTGTGTGCAGCCTGGAGTACTATCGCGCCGCGGTGGCGTACGTGGCCGCGCGTGTAGAGAAACCGGAATTTTTCGTCTTTACCGATGATCCGGACTGGACGCGTACCAAGCTCAAATTGGATTTTCCGGCCTACCTGGTTACCCAGAATCAGGACGCGCCTGTGGAAGATCTGCGTCTGATGACCCTGTGTCGTCATCATATTATCGCGAACTCTAGCTTTTCCTGGTGGGGTGCTTGGCTGGGCGAAAAGCCGGGTCAAATCGTTTGCGCACCGCAACGTTGGTTCGGCGCGTACCCGCACGATACCCGCGACCTGGTGCCGGATCGTTGGACACGATTGGACGGCTTA (SEQ ID NO:3).

[0023] (2) Helicobacter pylori-derived α-1,2-fucosyltransferase futC (WP_121037174.1), with codon optimization, and the specific nucleic acid sequence is shown below.

[0024] Nucleic acid sequence encoding the futC protein: ATGGCATTTAAGGTTGTTCAGATTTGCGGGGGGTTAGGGAATCAGATGTTTCAATATGCGTTTGCGAAAAGCCTGCAAAAACACTCAAATACGCCGGTTCTGCTGGATATTACGTCGTTTGATTGGTCAGATAGAAAAATGCAACTGGAACTGTTTCCGATCGATCTGCCATATGCAAGCGAAAAAGAAATTGCAATCGCAAAAATGCAACATCTGCCTAAACTGGTGAGAGATGCGCTGAAATGCATGGGATTCGACCGCGTTTCAAAAGAAATTGTTTTTGAATACGAGCCGGAACTGCTGAAACCGTCAAGACTGACATACTTCTATGGTTACTTCCAAGATCCGAGATATTTTGATGCGATCAGTCCTCTGATTAAACAAACATTTACACTGCCGCCGCCGCCGCCTGAAAATGGCAACAATAAAAAGAAAGAAGAGGAATATCACCGCAAATTAGCACTGATTCTGGCAGCAAAAAATTCAGTTTTTGTTCATATTAGAAGAGGCGATTATGTTGGCATTGGCTGCCAACTGGGCATTGATTATCAAAAAAAAGCACTGGAATATATGGCAAAAAGAGTTCCGAATATGGAACTGTTTGTTTTTTGCGAAGATCTGACATTTACACAAAATCTGGATCTGGGCTATCCGTTTATGGATATGACAACAAGAGATAAAGAAGAAGAAGCATATTGGGATATGCTGCTGATGCAATCATGCCAACATGGCATTATTGCAAATTCAACATATTCATGGTGGGCAGCATATCTGATTAATAATCCGGAAAAAATTATTATTGGCCCGAAACATTGGCTGTTTGGCCATGAAAATATTCTGTGCAAAGAATGGGTTAAAATTGAATCACATTTTGAAGTTAAATCACAAAAATATAATGCATAA (SEQ ID NO:4).

[0025] (3) Using plasmid pACYCDuet-1 as the expression vector backbone, respectively with the optimized ASfutC、futCUsing nucleic acid sequences as target fragments, pACYC-ASfutC expression vectors and pACYC-futC expression vectors were constructed to obtain stable expression cassettes at the plasmid level, which are formed by cis-linking the Ptac promoter with different α-1,2-fucosyltransferase genes.

[0026] Example 3 Construction of recombinant strains This embodiment employs a genome integration approach, utilizing the CRISPR / Cas9 genome editing system to preserve the chassis strain MG (with the β-galactosidase gene knocked out) in the laboratory. lacZ and UDP-glucose lipid transporter transferase gene wcaJ The dadX site of *E. coli* MG1655 is integrated with P... tac -ASfutC、P tac -futC was used to obtain recombinant Escherichia coli MG-ASfutC and MG-futC. The specific implementation method is as follows: (1) First, the pCas9 plasmid was transformed into the MG strain in the chassis, spread on a 50 μg / mL Kan plate, and incubated at 30℃ for 12 h. Then, single colonies were picked from the plate and inoculated into fresh LB medium. Kan antibiotic was added to a final concentration of 50 μg / mL, and the culture was incubated overnight at 30℃ and 220 r / min. The overnight culture was then transferred to a 250 mL LB medium containing 50 mL of LB medium at an inoculation rate of 1-2%. In a mL Erlenmeyer flask, when the bacterial cell OD600 reaches 0.2, arabinose at a final concentration of 30 mmol / L is added to induce the expression of λ-Red recombinase by the pCas9 plasmid. The culture is continued until the OD600 reaches 0.6-0.7. The bacterial culture is then incubated on ice for 20-30 min, followed by centrifugation at 5500 rpm to collect the bacterial cells. The cells are washed twice with pre-cooled sterile water, the supernatant is discarded, and the cells are resuspended in 400-500 μL of pre-cooled 10% glycerol to prepare MG competent cells carrying the pCas9 plasmid for later use.

[0027] (2) The N20 target sequence specific to the dadX site of E. coli MG1655 was screened using the CRISPR design platform (https: / / chopchop.cbu.uib.no / ), and the N20 sequence was cloned into the sgRNA expression backbone to construct the sgRNA expression vector pTarget-dadX for targeting the dadX site. The nucleotide sequence of pTarget-dadX is as follows:

[0028] (3) Using fusion PCR technology, primers for the upstream and downstream homologous arms of the dadX site in E. coli MG1655, as well as primers for Ptac-ASfutC and Ptac-futC, were first designed, and complementary overlapping sequences (about 20 bp) were introduced into the primers of adjacent fragments. Homologous arms of the dadX site and Ptac-ASfutC and Ptac-futC fragments were obtained by PCR amplification. During the amplification process, high-fidelity polymerase was used to ensure high fidelity and yield of the fragments. The amplified fragments were mixed and extended to form a complete DNA template by complementary pairing of the overlapping regions under fusion PCR conditions. Then, the outer primers were used for amplification to obtain the target integrated fragments HA-dadX-Ptac-ASfutC and HA-dadX-Ptac-futC.

[0029] (4) The above-mentioned integrated fragments HA-dadX-Ptac-ASfutC, HA-dadX-Ptac-futC, and pTarget-dadX were electroporated into E. coli MG competent cells containing the pCas9 plasmid, respectively. 800 μL of LB medium was added, and the cells were cultured at 30℃ and 220 rpm for 2 h. The cells were then plated on plates containing kanamycin (50 μg / mL) and spectinomycin (50 μg / mL) and cultured overnight at 30℃. Single colonies were picked for PCR verification and DNA sequencing verification to confirm that the exogenous α-1,2-fucosyltransferase genes ASfutC and futC were successfully integrated into the dadX integration site of E. coli. The pTarget and pCas9 plasmids were eliminated from the verified colonies. Single colonies were picked and cultured on LB medium containing IPTG (5 mmol / L) and kanamycin at 30℃ and 220 rpm for 12 h to induce the elimination of the pTarget plasmid. The bacterial culture was streaked onto LB agar plates containing kanamycin and incubated overnight at 30°C. Single colonies were then spotted onto plates containing kanamycin and spectinomycin, respectively. If the colonies grew on the kanamycin-containing plate but not on the spectinomycin-containing plate, the pTarget plasmid was successfully eliminated. pCas9 is a temperature-sensitive plasmid. Single colonies with successfully eliminated pTarget plasmids were picked and incubated overnight at 37°C on antibiotic-free LB agar plates to eliminate the pCas9 plasmid. The bacterial culture was streaked onto antibiotic-free LB agar plates and incubated at 37°C. Single colonies were then spotted onto LB agar plates containing kanamycin and antibiotic-free plates, respectively. If the colonies grew on the antibiotic-free plate but not on the kanamycin-containing plate, the pCas9 plasmid was eliminated, resulting in plasmid-free recombinant Escherichia coli MG-ASfutC and MG-futC.

[0030] Example 3: Fermentation Validation of Recombinant Strains (1) The recombinant Escherichia coli MG-ASfutC and MG-futC obtained in Example 2 were fermented and verified. The specific operation was as follows: single colonies on fresh plates were picked and placed into seed culture medium and cultured for 10 h to obtain seed liquid; the seed liquid was inoculated into fermentation culture medium at an inoculation rate of 1% and cultured at 30℃ and 220 rpm for 72 h to obtain fermentation broth.

[0031] The seed culture medium contains the following components: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl. The fermentation culture medium contains the following components: 4 g / L glycerol, 12 g / L tryptone, 24 g / L yeast extract, 12.54 g / L dipotassium hydrogen phosphate, 2.31 g / L potassium dihydrogen phosphate, and 20 g / L lactose.

[0032] (2) The synthesis of 2'-FL and the production of DFL in the fermentation broth of recombinant *E. coli* were detected using a high-performance liquid chromatography (HPLC) system. The specific operation was as follows: The concentration of 2'-FL in the fermentation supernatant was determined using an Aminex HPX-87H organic acid column (300*7.8mm). The mobile phase of the HPLC was 5mM H2SO4, the detector was a differential detector, the column detection temperature was set to 60℃, and the detection flow rate was set to 0.6mL / min. After centrifuging 2mL of fermentation broth at 12000rpm for 15min, the supernatant was collected, filtered through a filter membrane, and then used for HPLC analysis to determine the content of 2'-FL and DFL in the fermentation broth. The HPLC chromatogram results were obtained as shown below. Figure 2 As shown, the contents of 2'-FL and DFL in the fermentation broth of different recombinant strains are as follows: Figure 3 As shown.

[0033] The test results showed that the yield of 2'-FL synthesized de novo by recombinant Escherichia coli MG-ASfutC reached 7.9 g / L, which is 3.1 times the yield of recombinant Escherichia coli MG-futC. Moreover, no DFL was produced during the fermentation of recombinant Escherichia coli MG-ASfutC.

[0034] Example 4: Scale-up fermentation experiment of recombinant strain To further verify the production effect of the strain MG-ASfutC constructed in Example 2 in a 5L fermenter, this experiment inoculated the MG-ASfutC seed culture into a 5L fermenter containing 1.8L of fermentation medium at a 10% (v / v) inoculation ratio. The culture conditions were: dissolved oxygen set at 35%, growth temperature at 37℃, automatic adjustment of stirring speed to maintain dissolved oxygen level, aeration rate of 2.5 vvm, and pH set at 6.7 ± 0.1. During fermentation, ammonia was used to adjust the pH value to ensure pH stability throughout the fermentation process. To accurately control the substrate concentration, the consumption of lactose and glycerol was monitored periodically, and lactose and glycerol were replenished as needed to maintain a stable lactose concentration of 10 g / L in the fermentation system, while ensuring that the glycerol concentration remained below 1 g / L.

[0035] After scaled-up fermentation, the product components were analyzed, and the results are as follows: Figure 4 As shown in the figure, the final yield of 2'-FL reached 73.3 g / L, with a production intensity of 1.22 g / L / h. Calculations showed a carbon source conversion rate of 0.51 g / g glycerol and a lactose conversion rate of 1.22 g / g lactose, indicating a high substrate conversion efficiency in this fermentation system. These results validate the feasibility and production potential of the recombinant strain MG-ASfutC under large-scale fermentation conditions, providing strong support for industrial application.

[0036] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A recombinant *Escherichia coli* strain for producing 2'-fucosylated lactose, characterized in that, Recombinant Escherichia coli genome integration and overexpression ASfutC Gene, ASfutC The nucleic acid sequence of the gene is shown as SED ID NO:2 or SED ID NO:

3.

2. The recombinant Escherichia coli as described in claim 1, characterized in that, Recombinant E. coli to knock out the β-galactosidase gene lacZ and UDP-glucose lipid transporter transferase gene wcaJ The Escherichia coli MG1655 was the chassis strain.

3. The recombinant Escherichia coli as described in claim 1, characterized in that, ASfutC The integration site of the gene is the dadX site in E. coli MG1655.

4. The recombinant Escherichia coli as described in claim 1, characterized in that, Recombinant E. coli utilizes the promoter tac to ASfutC The gene is overexpressed.

5. The recombinant Escherichia coli as described in claim 1, characterized in that, The integration was performed using the CRISPR / Cas9 genome editing system.

6. The use of the recombinant Escherichia coli as described in any one of claims 1-5 in the preparation of 2'-fucosylated lactose.

7. A method for producing 2'-fucosylated lactose, characterized in that, Using the recombinant Escherichia coli as described in any one of claims 1-5 as the fermentation strain, 2'-fucosylated lactose is produced in a fermentation system with glycerol and lactose as substrates.

8. The method as described in claim 7, characterized in that, The fermentation system includes the following components in the following amounts: glycerol 4 g / L, tryptone 12 g / L, yeast extract 24 g / L, dipotassium hydrogen phosphate 12.54 g / L, potassium dihydrogen phosphate 2.31 g / L, and lactose 20 g / L.

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