Engineered strain for high-yield 2'-fucosyllactose and construction method and application thereof

Abstract

The application belongs to the technical field of biology and relates to an engineered strain for high-yield 2'-fucosyllactose and a construction method and application thereof. Metabolic engineering is used to make Yarrowia lipolytica have the ability to absorb lactose and synthesize GDP-L-fucose in cells, and the cell can increase the yield of 2'-fucosyllactose by expressing a Ub-tagged alpha-1,2-fucosyltransferase, and then the GDP-D-mannose synthesis pathway and the NADPH regeneration pathway are enhanced, so that the obtained recombinant Yarrowia lipolytica strain has the ability of high-yield 2'-fucosyllactose.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to engineered strains that produce high levels of 2'-fucosylated lactose, their construction methods, and applications. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] The biosynthesis of 2'-fucosylated lactose currently takes place primarily in *Escherichia coli*. Through a series of optimizations, the yield of 2'-fucosylated lactose in *E. coli* has become quite substantial. However, because *E. coli* is a pathogenic bacterium, it may be contaminated with endotoxins, posing certain safety risks. Furthermore, *E. coli* is susceptible to phage contamination during fermentation, thus limiting large-scale industrial production.

[0004] Yarrowia lipolytica, generally considered a safe microorganism, is widely used in the production of various food compounds. Previous studies have shown that Yarrowia lipolytica can be used for the synthesis of 2'-fucosylated lactose; however, the current yield of 2'-fucosylated lactose produced using Yarrowia lipolytica is only 24 g / L, far lower than that of other microorganisms. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an engineered strain that produces high levels of 2'-fucosylated lactose, along with its construction method and applications. Through metabolic engineering, *Yarrowia lipolytica* is enabled to absorb lactose and synthesize GDP-L-fucose intracellularly. Furthermore, by expressing an α-1,2-fucosyltransferase fused with a Ub tag, the cell can increase its 2'-fucosylated lactose production. Subsequently, by enhancing the GDP-D-mannose synthesis pathway and the NADPH regeneration pathway, the resulting recombinant *Yarrowia lipolytica* strain possesses the ability to produce high levels of 2'-fucosylated lactose.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] On one hand, an engineered strain that produces high levels of 2'-fucosylated lactose, originating from *Yarrowia lipolytica*, expresses lactose transporter, GDP-mannose dehydratase, GDP-fucosylated enzyme, and α-1,2-fucosylated transferase fused with a Ub tag. This strain enhances the GDP-D-mannose synthesis pathway by overexpressing 6-phosphate mannose isomerase, phosphate mannose mutase, and 1-phosphate mannose guanylate transferase using a strong promoter. Furthermore, it enhances the NADPH regeneration pathway by overexpressing 6-phosphate glucose dehydrogenase, 6-phosphate gluconolactonease, and 6-phosphate gluconolactone dehydrogenase using a strong promoter.

[0008] On the other hand, a method for constructing an engineered strain that produces high levels of 2'-fucosylated lactose includes the following steps:

[0009] An integrative plasmid containing the expression cassettes of the lactose transporter gene klLAC12, the GDP-mannose dehydratase gene maGMD, the GDP-fucose synthase gene maGMER, and the α-1,2-fucosyltransferase gene Ub-futC fused with a Ub tag was constructed and linearized. The linearized plasmid was then integrated into the genome of Yarrowia lipolytica PO1f using an iterative integration method to obtain the first recombinant Yarrowia lipolytica strain.

[0010] An integrated plasmid containing expression cassettes encoding 6-phosphoglucono isomerase PMI40, phosphoglucono isomerase SEC53, and 1-phosphoglucono guanylate transferase PSA1 was constructed and linearized, and transformed into the first recombinant Yersinia lipolytica strain to obtain the second recombinant Yersinia lipolytica strain.

[0011] An integrated plasmid containing expression cassettes encoding glucose-6-phosphate dehydrogenase gene ZWF1, gluconolactone-6-phosphate esterase gene PGL, and gluconolactone-6-phosphate dehydrogenase gene GND1 was constructed, linearized, and transformed into a second recombinant Yersinia lipolytica strain.

[0012] Thirdly, the application of an engineered strain that produces high levels of 2'-fucosylated lactose in the production of 2'-fucosylated lactose.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] This invention utilizes metabolic modification to enable the *Yarrowia lipolytica* strain to absorb lactose and synthesize GDP-L-fucose intracellularly using GDP-D-mannose as a precursor. Furthermore, by heterologously expressing α-1,2-fucosyltransferase fused with a Ub tag, the strain achieves a higher yield of 2'-fucosyllactose than strains expressing α-1,2-fucosyltransferase without the Ub tag. Subsequently, through iterative integration of the 2'-FL synthesis pathway, enhancement of the GDP-D-mannose synthesis pathway, and NADPH regeneration pathway, the yield of 2'-FL is significantly increased, resulting in the construction of a high-yielding strain. After fermentation, the strain achieved a 2'-FL yield of 41.10 g / L, the highest known yield among yeasts, demonstrating significant industrial application value and representing an advanced level in similar technologies. Attached Figure Description

[0015] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0016] Figure 1 This is a graph showing the yield of 2'-FL from eight transformants of the F1 series strain in the embodiment of the present invention.

[0017] Figure 2 This is a graph showing the yield of 2'-FL from eight transformants of the F2 series strain in the embodiment of the present invention.

[0018] Figure 3 This is a graph showing the yield of 2'-FL from eight transformants of the F3 series strain in the embodiment of the present invention.

[0019] Figure 4 This is a graph showing the yield of 2'-FL from eight transformants of the F4 series strain in the embodiment of the present invention.

[0020] Figure 5 This is a graph showing the yield of 2'-FL from eight transformants of the F5 series strain in the embodiment of the present invention.

[0021] Figure 6 This is a diagram showing the yield of 2'-FL produced by the engineered strain F5-1 during fermentation in an embodiment of the present invention. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0024] Given the current low yield of 2'-fucosylated lactose by Yersinia lipolytica, this invention proposes an engineered strain for high 2'-fucosylated lactose production, its construction method, and its application.

[0025] In one typical embodiment of the present invention, an engineered strain that produces high levels of 2'-fucosylated lactose is provided. The originating strain of the engineered strain is *Yarrowia lipolytica*. The engineered strain can express lactose transporter, GDP-mannose dehydratase, GDP-fucosylated enzyme, and α-1,2-fucosylated transferase fused with a Ub tag. The engineered strain can enhance the GDP-D-mannose synthesis pathway by overexpressing 6-phosphate mannose isomerase, phosphate mannose mutase, and 1-phosphate mannose guanylate transferase using a strong promoter. The engineered strain can enhance the NADPH regeneration pathway by overexpressing 6-phosphate glucose dehydrogenase, 6-phosphate gluconolactonease, and 6-phosphate gluconolactone dehydrogenase using a strong promoter.

[0026] The genes encoding lactose transporter, GDP-mannose dehydratase, and GDP-fucosylate synthase in the engineered strain of this invention are klLAC12, maGMD, and maGMER, respectively, and their DNA sequences are referenced from those described in Hollands, K., et al. Engineering two species of yeast as cell factories for 2'-fucosyllactose. Metab Eng, 2019.

[0027] In some embodiments, in α-1,2-fucosyltransferases fused with a Ub tag, the Ub tag is fused to the N-terminus of the α-1,2-fucosyltransferase. The α-1,2-fucosyltransferase is derived from Helicobacter pylori 26695.

[0028] In some embodiments, the gene encoding the α-1,2-fucosyltransferase fused with the Ub tag is shown in SEQ ID NO.1, and the DNA sequence of the gene Ub-futC is as shown in SEQ ID NO.1.

[0029] In some embodiments, the gene encoding 6-phosphate mannose isomerase is the PMI40 gene, with the gene number YALI0_B18348g.

[0030] In some embodiments, the gene encoding mannose phosphate mutase is the SEC53 gene, with the gene number YALI0_D13112g.

[0031] In some embodiments, the gene encoding 1-phospho-mannose-guanylate transferase is the PSA1 gene, with the gene number YALI0_C06490g.

[0032] In some embodiments, the gene encoding glucose-6-phosphate dehydrogenase is the ZWF1 gene, with the gene number YALI0_E22649g.

[0033] In some embodiments, the gene encoding 6-phosphogluconide lactonease is the PGL gene, with the gene number YALI0_C19085g.

[0034] In some embodiments, the gene encoding 6-phosphoglucate dehydrogenase is the GND1 gene, with the gene number YALI0_B15598g.

[0035] Another embodiment of the present invention provides a method for constructing the above-mentioned engineered strain that produces high levels of 2'-fucosylated lactose, comprising the following steps:

[0036] An integrative plasmid containing the expression cassettes of the lactose transporter gene klLAC12, the GDP-mannose dehydratase gene maGMD, the GDP-fucose synthase gene maGMER, and the α-1,2-fucosyltransferase gene Ub-futC fused with a Ub tag was constructed and linearized. The linearized plasmid was then integrated into the genome of Yarrowia lipolytica PO1f using an iterative integration method to obtain the first recombinant Yarrowia lipolytica strain.

[0037] An integrated plasmid containing expression cassettes encoding 6-phosphoglucono isomerase PMI40, phosphoglucono isomerase SEC53, and 1-phosphoglucono guanylate transferase PSA1 was constructed and linearized, and transformed into the first recombinant Yersinia lipolytica strain to obtain the second recombinant Yersinia lipolytica strain.

[0038] An integrated plasmid containing expression cassettes encoding glucose-6-phosphate dehydrogenase gene ZWF1, gluconolactone-6-phosphate esterase gene PGL, and gluconolactone-6-phosphate dehydrogenase gene GND1 was constructed, linearized, and transformed into a second recombinant Yersinia lipolytica strain.

[0039] In some embodiments, the linearized pULUF-GG plasmid is transformed into a *Yarrowia lipolytica* strain, allowing it to integrate into the genome of the *Yarrowia lipolytica* strain to obtain a first pre-recombinant *Yarrowia lipolytica* strain. The Ura3 gene expression cassette is removed from the pre-recombinant *Yarrowia lipolytica* strain. Then, the linearized pULUF-GG plasmid is transformed into the first pre-recombinant *Yarrowia lipolytica* strain with the Ura3 gene expression cassette removed to obtain a second pre-recombinant *Yarrowia lipolytica* strain. Finally, the linearized pULUF-GG plasmid is transformed into the second pre-recombinant *Yarrowia lipolytica* strain. The first recombinant *Yersinia lipolytica* strain was obtained from recombinant *Yersinia lipolytica*. Among them, the pULUF-GG plasmid is an integrated plasmid containing the expression cassette of the lactose transporter gene klLAC12, the expression cassette of the GDP-mannose dehydratase gene maGMD, the expression cassette of the GDP-fucose synthase gene maGMER, and the expression cassette of the α-1,2-fucosyltransferase gene Ub-futC fused with the Ub tag; the pHLUF-GG plasmid is the pULUF-GG plasmid with the Ura3 gene replaced by the hyg gene.

[0040] Specifically, the futC and Ub genes were fused using fusion PCR to construct the fusion protein-coding gene Ub-futC. Then, the klLAC12, maGMD, maGMER, and Ub-futC genes, along with their respective gene regulators' promoters and terminators, were fused using fusion PCR to construct the klLAC12, maGMD, maGMER, and Ub-futC gene expression cassettes, respectively. These cassettes were then ligated into a vector backbone labeled with the Ura3 gene expression cassette using Gibson assembly to obtain the pULUF-GG plasmid. The primer sets for amplifying the maGMD gene are shown in SEQ ID NO.2 and SEQ ID NO.3. The primer sets for amplifying the maGMER gene are shown in SEQ ID NO.4 and SEQ ID NO.5. The primer sets for amplifying the futC gene are shown in SEQ ID NO.6 and SEQ ID NO.7. The primer sets for amplifying the Ub gene are shown in SEQ ID NO. 8 and SEQ ID NO. 9. The primer sets for amplifying the klLAC12 gene are shown in SEQ ID NO. 10 and SEQ ID NO. 11.

[0041] In some embodiments, a second recombinant Yersinia lipolytica strain is obtained by transforming a linearized pMAN plasmid into a first recombinant Yersinia lipolytica strain; wherein the pMAN plasmid is an integrated plasmid containing an expression cassette encoding the 6-phosphate mannose isomerase gene PMI40, the phosphate mannose mutase gene SEC53, and the 1-phosphate mannose guanylate transferase gene PSA1.

[0042] Specifically, the PMI40 gene, SEC53 gene, and PSA1 gene, along with their respective gene regulators' promoters and terminators, were constructed using fusion PCR to create the PMI40 gene expression cassette, SEC53 gene expression cassette, and PSA1 gene expression cassette. These cassettes were then ligated into a vector backbone labeled with the leu gene expression cassette using Gibson assembly to obtain the pMAN plasmid. The primer sets for amplifying the pmi40 gene are shown in SEQ ID NO. 12 and SEQ ID NO. 13. The primer sets for amplifying the SEC53 gene are shown in SEQ ID NO. 14 and SEQ ID NO. 15. The primer sets for amplifying the psa1 gene are shown in SEQ ID NO. 16 and SEQ ID NO. 17.

[0043] In some embodiments, the linearized pZPG plasmid is transformed into a second recombinant Yersinia lipolytica strain with the Trp gene knocked out to obtain an engineered strain; wherein the pZPG plasmid is an integrated plasmid containing expression cassettes of the glucose-6-phosphate dehydrogenase encoding gene ZWF1, the gluconolactone-6-phosphate enzyme encoding gene PGL, and the gluconolactone-6-phosphate dehydrogenase encoding gene GND1.

[0044] Specifically, the ZWF1 gene, PGL gene, and GND1 gene, along with their respective gene-regulated promoters and terminators, were constructed using fusion PCR to create the ZWF1 gene expression cassette, PGL gene expression cassette, and GND1 gene expression cassette, respectively. These cassettes were then ligated into a vector backbone containing the Trp gene expression cassette using Gibson assembly to obtain the pZPG plasmid. The primer sets for amplifying the ZWF1 gene are shown in SEQ ID NO. 18 and SEQ ID NO. 19. The primer sets for amplifying the PGL gene are shown in SEQ ID NO. 20 and SEQ ID NO. 21. The primer sets for amplifying the GND1 gene are shown in SEQ ID NO. 22 and SEQ ID NO. 23.

[0045] A third embodiment of the present invention provides the application of the above-mentioned engineered strain that produces high levels of 2'-fucosylated lactose in the production of 2'-fucosylated lactose.

[0046] Specifically, an engineered strain that produces high levels of 2'-fucosylated lactose was activated and inoculated into a seed culture medium. The resulting seed culture was then inoculated into a fermentation medium, and 2'-fucosylated lactose was obtained through fermentation.

[0047] More specifically, the seed culture inoculation amount is 1–10% (V / V).

[0048] More specifically, the seed culture medium contains 1.5–2.5% glucose, 1.5–2.5% tryptone, and 0.5–1.5% yeast extract.

[0049] More specifically, the fermentation medium contains 6-8% glucose, 2-4% tryptone, 1-2% yeast extract, and 0.4-1% lactose.

[0050] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0051] Example 1

[0052] I. Materials and Methods:

[0053] 1. In this embodiment, gene synthesis, primer synthesis, and sequencing were completed by Beijing Qingke Xinyue Biotechnology Co., Ltd.

[0054] 2. Unless otherwise specified, the experimental methods used in this embodiment, including plasmid construction, enzyme digestion, preparation of competent cells, and transformation, are all conventional methods. Specific experimental conditions can be determined through simple experiments if necessary.

[0055] 3. Unless otherwise specified, all materials and reagents used in this embodiment can be obtained commercially.

[0056] 4. The original strain of Yarrowia lipolytica involved in this embodiment is PO1f (ATCC number MYA-2613; genotype...).

[0057] MATAura3-3021leu2-270xpr2-322axp2-deltaNU49XPR2::SUC2) was purchased from ATCC.

[0058] 5. The genes involved in this embodiment, namely GDP-mannose dehydratase (GMD) and GDP-fucose synthase (GMER) from *Morchella alpina*, lactose transporter (LAC12) from *Kluyveromyces lactis*, and fucosyltransferase (FutC) from *Helicobacter pylori*, were all obtained through gene synthesis.

[0059] 6. LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15

[0060] g / L agar powder.

[0061] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0062] YPD medium: 1% yeast extract, 2% tryptone, 2% glucose.

[0063] YPD solid medium: 1% yeast extract, 2% tryptone, 2% glucose, 2% agar powder.

[0064] II. Preparation of the target gene:

[0065] 1. Based on the sequence described in Hollands, K., et al. Engineering two species of yeast as cellfactories for 2'-fucosyllactose. Metab Eng, 2019., Beijing Qingke Xinyue Biotechnology Co., Ltd. was commissioned to synthesize the optimized lactose transporter (LAC12) encoding gene klLac12, GDP-mannose dehydratase (GMD) gene maGMD, and GDP-fucosylate synthase (GMER) gene maGMER.

[0066] 2. Based on the nucleotide sequence of α-1,2-fucosyllactose (FutC) from Helicobacter pylori described in Hollands, K., et al., Engineering two species of yeast as cellfactories for 2'-fucosyllactose. Metab Eng, 2019, after codon optimization, the optimized fucosyllactose gene futC was synthesized by Beijing Qingke Xinyue Biotechnology Co., Ltd. The optimized Ub tag was then fused to the N-terminus of FutC to construct the Ub-futC gene. The nucleotide sequence of Ub-futC is shown in SEQ ID NO.1.

[0067] 4. Endogenous genes including PMI40, SEC53, PSA1 and PGL were obtained by PCR amplification using the genome of strain PO1f as a template; ZWF1 and GND1 were obtained by PCR amplification using the cDNA of strain PO1f as a template.

[0068] 5. The Ub-futC sequence is as follows:

[0069] >SEQ ID NO.1

[0070]

[0071] The primer sequences are shown in Table 1.

[0072] Table 1 Primer sequences

[0073]

[0074]

[0075]

[0076] III. Plasmid Construction:

[0077] 1. Construction of plasmids pLEP-futC-GGL and pLEP-Ub-futC-GGL

[0078] The plasmid pLEP-futC-GGL was constructed primarily via fusion PCR, amplifying the klLAC12, maGMD, and maGMER genes, along with their promoters and terminators for regulation. Expression cassettes of these genes were then obtained via fusion PCR. Using the pLEP-leu plasmid as a backbone, the futC gene was cloned into the UT8 promoter of the pLEP-leu plasmid via conventional Gibson assembly. Subsequently, the expression cassettes of the klLAC12, maGMD, and maGMER genes were cloned into the pLEP-futC plasmid via Gibson assembly.

[0079] The plasmid pLEP-Ub-futC-GGL was constructed by first amplifying the optimized Ub tag and futC gene separately via PCR, and then fusing the two genes using fusion PCR. The resulting fusion gene was then cloned into the pLEP-leu plasmid using Gibson assembly to obtain the pLEP-Ub-futC plasmid. Subsequently, the klLAC12, maGMD, and maGMER gene expression cassettes were also cloned into the pLEP-Ub-futC plasmid.

[0080] 2. Construction of plasmid pULUF-GG

[0081] The plasmid pULUF-GG was mainly constructed via fusion PCR. First, the futC and Ub genes were used to construct the fusion protein encoding gene using fusion PCR. Then, the klLAC12, maGMD, and maGMER genes, along with the promoters and terminators for regulating these genes, were amplified. The expression cassettes of these genes were obtained by fusion PCR, and a vector backbone containing the Ura3 gene expression cassette, which contains LoxP sites upstream and downstream, was amplified. Subsequently, the gene expression cassettes were ligated together using Gibson assembly to obtain the pULUF-GG plasmid.

[0082] 3. Construction of plasmid pHLUF-GG

[0083] The plasmid pHLUF-GG was obtained by replacing the Ura3 selection marker gene in the pULUF-GG plasmid with the hyg gene. Primers were designed using the pULUF-GG plasmid as a template to amplify the fragment that does not contain the Ura3 selection marker gene expression cassette, while amplifying the expression cassette containing the hyg gene. The pHLUF-GG plasmid was then constructed by Gibson assembly.

[0084] 4. Construction of plasmid pMAN

[0085] Using the genome of strain PO1f as a template, the PMI40, SEC53 and PSA1 genes were amplified, and the promoters and terminators used to regulate the expression of the above genes were amplified, respectively. The expression cassettes of the above genes were constructed by fusion PCR, and the vector backbone containing the leu gene expression cassette was amplified at the same time. Finally, the pMAN plasmid was constructed by Gibson assembly.

[0086] 5. Construction of plasmids overexpressing ZWF1, PGL, and GND1 genes

[0087] Using pKi-1 plasmid as the starting plasmid, the leu gene on the plasmid was replaced with the Trp gene by PCR and Gibson assembly. The ZWF1 gene, PGL gene and GND1 gene were amplified and constructed at the Bsp119I restriction site of the plasmid to construct free expression plasmids pTrp-ZWF1, pTrp-PGL and pTrp-GND1.

[0088] 6. Construction of plasmid pZPG

[0089] Using the genome of strain PO1f as a template, the ZWF1 gene, PGL gene, and GND1 gene were amplified, and the promoters and terminators used to regulate the expression of the above genes were amplified. The expression cassettes of the above genes were constructed by fusion PCR, and the vector backbone containing the Trp gene expression cassette was amplified at the same time. Finally, the pZPG plasmid was constructed by Gibson assembly.

[0090] Example 2: Construction of an engineered Yarrowia lipolytica strain that produces high levels of 2'-fucosylated lactose.

[0091] 1. Construct a Yarrowia lipolytica strain capable of producing 2'-fucosylated lactose. Transform plasmids pLEP-futC-GGL and pLEP-Ub-futC-GGL into Yarrowia lipolytica strain PO1f, enabling Yarrowia lipolytica to synthesize GDP-L-fucosylated lactose intracellularly and transport lactose into the cell, thus enabling the synthesis of 2'-FL. The specific method is as follows: (1) Yarrowia lipolytica po1f was cultured overnight in YPD liquid medium (containing 2% tryptone, 1% yeast extract and 2% glucose), and competent cells were prepared using the conventional method for preparing competent yeast lithium acetate cells. (2) 1 μL (free plasmid) / 10 μL (linearized plasmid) plasmid was added to 40 μL of competent cells, followed by 2 μL of salmon sperm DNA, and the cells were incubated in a 30℃ water bath for 15 min. (3) Add 350 μL of PEG 4000-Lithium Acetate (0.1M pH 6.0) and 16 μL of 1M DTT (40mM) to the above system, and incubate statically in a water bath at 30°C for 1 h. (4) Add 40 μL of DMSO (final concentration approximately 40%) to the above system, and heat shock at 39°C for 10 min. (5) Add 600 μL of Lithium Acetate (0.1M pH 6.0), and incubate at room temperature for 15 min. (6) Spread 200 μL of the above mixture onto an SD-ura+leu screening plate and incubate at 30°C for 2-3 days. (7) Randomly select transformants and detect 2'-fucosylated lactose in the transformants. Transformants and the original strain were fermented separately in YPD liquid (80 g / L glucose) medium supplemented with 0.5% lactose. After 144 h, the fermentation broth was collected for analysis. The analysis was performed using a Rezex ROA Organic Acid H+ (8%) column via high-performance liquid chromatography (HPLC). The HPLC instrument was purchased from Shimadzu Corporation. Specific conditions were: 5 mM sulfuric acid as the mobile phase, column temperature set at 65 °C, flow rate at 0.6 mL / min, and injection volume at 10 μL. The intracellular 2'-FL yield was determined by centrifugation at 12000 rpm, washing twice with distilled water, followed by high-speed grinding and lysis. The intracellular 2'-FL yield was then measured by analyzing the resulting cell lysates. The results showed that the 2'-FL yield in the pLEP-futC-GGL transformed strain was 0.10 g / L, while the 2'-FL yield in the pLEP-Ub-futC-GGL transformed strain reached 0.23 g / L, indicating that the expression level of FutC fused with the Ub tag was increased, thereby promoting the accumulation of 2'-FL.

[0092] 2. A *Yersinia lipolytica* strain capable of stably producing 2'-fucosylated lactose was constructed. The linearized pULUF-GG plasmid was transformed into *Yersinia lipolytica* strain PO1f, integrating it into the PO1f genome. The specific transformation method was the same as described above. Results showed that 2'-FL was detected in the fermentation broth of the transformed strains, with the recombinant strain F1-4 achieving a yield of 0.71 g / L. Figure 1 As shown. The original strain PO1f did not produce 2'-fucosylated lactose.

[0093] 3. Using recombinant *Yarrowia lipolytica* strain F1-4 as the starting strain, the Ura3 gene expression cassette was removed using Cre recombinase. The linearized pULUF-GG plasmid was then transformed again into the F1-4 strain after the Ura3 gene expression cassette was removed. The specific transformation method was the same as described above. Transformants were picked and fermented in YPD liquid medium. After 144 hours, 2'-FL was detected. The 2'-fucosylated lactose detection method was the same as described above. The results showed that the 2'-FL yield in the transformant strains was higher than that in the F1-4 strain, with the highest 2'-FL yield in the F2-4 strain, reaching 0.85 g / L. Figure 2 As shown.

[0094] 4. Using recombinant *Yarrowia lipolytica* strain F2-4 as the starting strain, the linearized pHLUF-GG plasmid was transformed into the F2-4 strain to construct the F3 batch strain. The specific transformation method is the same as described above. Transformants were picked and fermented in YPD liquid medium. After 144 h, 2'-FL was detected. The 2'-fucosylated lactose detection method is the same as described above. The results showed that the 2'-FL yield in the F3 batch transformant strains was higher than that in the F2-4 strain, with the highest 2'-FL yield in the F3-5 strain, reaching 1.13 g / L. Figure 3 As shown.

[0095] 5. The synthesis of 2'-FL requires GDP-L-fucose as a direct precursor, which in turn is derived from GDP-D-mannose via two enzyme catalysis processes. Using recombinant *Yarrowia lipolytica* strain F3-5 as the starting strain, the linearized pMAN plasmid was transformed into F3-5 to enhance the supply of the precursor GDP-D-mannose, thereby promoting the accumulation of GDP-L-fucose and ultimately promoting the synthesis of 2'-FL. The specific transformation method is as described above. Transformants were selected and fermented in YPD liquid medium. After 144 hours, 2'-FL was detected. The 2'-FL detection method is as described above. The results showed that strain F4-2 had the highest 2'-FL yield, reaching 1.84 g / L. Figure 4 As shown.

[0096] 6. Using recombinant *Yarrowia lipolyticis* strain F4-2 as the starting strain, the Trp gene was first knocked out of the genome using CRISPR / Cas9. Then, plasmids pTrp-ZWF1, pTrp-PGL, and pTrp-GND1 were transformed into the resulting Trp knockout strains. Transformants were selected and fermented in SD minimal-Trp medium for verification. The results showed that the 2'-FL yield in the pTrp-PGL transformant was significantly higher than that in the F4-2 strain, indicating that pgl overexpression significantly promotes 2'-FL accumulation. Although the 2'-FL yield in the pTrp-ZWF1 and pTrp-GND1 transformants was lower than that in the F4-2 strain, their glucose conversion rate was significantly higher. Therefore, all three genes can promote 2'-FL accumulation.

[0097] 7. NADPH plays a crucial role in the 2'-FL synthesis pathway. To balance intracellular NADPH levels, the linearized pZPG plasmid was transformed into the F3-5 strain (Trp gene knocked out) to enhance the NADPH regeneration pathway. The specific transformation method is as described above. Transformants were picked and fermented in YPD liquid medium. After 144 hours, 2'-FL was detected. The 2'-fucosylated lactose detection method is as described above. The results showed that the F5-1 strain had the highest 2'-FL yield, reaching 2.87 g / L. Figure 5 As shown.

[0098] Example 3: Continuous fed-batch fermentation culture of strain F5-1

[0099] The recombinant strain F5-1, which produced high levels of 2'-FL as described in Example 2, was subjected to fed-batch fermentation in a 5L fermenter. The culture medium used in this example was 2X YPD medium (4% tryptone, 2% yeast extract, 7% glucose), with an initial volume of 3.6L. The bacterial culture was taken from the glycerol culture tube and streaked onto YPD agar plates, and cultured for 24 hours. The resulting single colonies were inoculated into a shake flask containing 50mL of YPD medium and cultured at 30°C and 220rpm for 24 hours. This seed culture was then transferred to a shake flask containing 200mL of YPD medium and cultured for another 24 hours. Finally, this seed culture was inoculated into the 5L fermenter. The fermenter was set to a temperature of 30°C, an aeration rate of 2.0 v / v / m, dissolved oxygen of 20%, and a stirring speed (800-1000 rpm) coupled to dissolved oxygen. The pH was maintained at 5.5. Glucose concentration in the fermenter was sampled and tested every 6 hours. Once the initial glucose in the culture medium was depleted, glucose was added continuously to maintain a very low glucose concentration in the medium. OD... 600When the biomass reaches about 85%, lactose is added in batches. Samples are taken every 6 hours to test the biomass, lactose consumption, and 2'-FL production in the fermenter. Figure 6 The fermentation results showed that the yield of 2'-FL reached its highest level of 41.10 g / L after 144 hours of fermentation.

[0100] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An engineered strain producing 2'-fucosylated lactose, characterized in that, The origin of the engineered strain is Yersinia lipolytica. The engineered strain overexpresses lactose transporter LAC12 from Kluyveromyces lactis, GDP-mannose dehydratase and GDP-fucose synthase from Morphyra alpineis, and α-1,2-fucosyltransferase fused with a Ub tag from Helicobacter pylori. The engineered strain also enhances the GDP-D-mannose synthesis pathway by overexpressing 6-phosphate mannose isomerase, phosphate mannose mutase and 1-phosphate mannose guanylate transferase by using a strong promoter, and enhances the NADPH regeneration pathway by overexpressing 6-phosphate glucose dehydrogenase, 6-phosphate gluconolactonease and 6-phosphate gluconolactone dehydrogenase by using a strong promoter. The *Yersinia lipolytica* is *Yersinia lipolytica* (…). Yarrowia lipolytica PO1f, ATCC number MYA-2613; In α-1,2-fucosyltransferases fused with a Ub tag, the Ub tag is fused to the N-terminus of the α-1,2-fucosyltransferase; the gene encoding the Ub-tagged α-1,2-fucosyltransferase is [gene name missing]. Ub-futC Its DNA sequence is shown in SEQ ID NO.1; The gene encoding 6-phosphate mannose isomerase is: PMI40 The gene, with gene number YALI0_B18348g; The gene encoding phosphomannose mutase is SEC53 The gene, with gene number YALI0_D13112g; The gene encoding 1-phosphomannose-guanylate transferase is: PSA1 The gene, with gene number YALI0_C06490g; The gene encoding glucose-6-phosphate dehydrogenase is: ZWF1 The gene, with gene number YALI0_E22649g; The gene encoding 6-phosphogluconide lactonease is: PGL The gene, with gene number YALI0_C19085g; The gene encoding 6-phosphoglucate dehydrogenase is: GND1 The gene, with the gene number YALI0_B15598g.

2. A method for constructing an engineered strain producing 2'-fucosylated lactose as described in claim 1, characterized in that, Includes the following steps: Constructing a gene containing lactose transporter klLAC12 Expression cassette, GDP-mannose dehydrase gene maGMD Expression cassette, GDP-fucosylation gene maGMER The expression cassette and the α-1,2-fucosyltransferase gene fused with the Ub tag Ub-futC The expression frame was integrated into a linearized plasmid and then integrated into *Yersinia lipophila* using an iterative integration method. Yarrowia lipolytica The first recombinant Yersinia lipophila strain was obtained from the genome of PO1f; Construct a gene encoding mannose-6-phosphate isomerase. PMI40 Phosphomannose mutase encoding gene SEC53 and the gene encoding 1-phosphoguanosine monophosphate transferase PSA1 The expression cassette was integrated into a plasmid and linearized, and then transformed into the first recombinant Yersinia lipophila strain to obtain the second recombinant Yersinia lipophila strain. Constructing a gene encoding glucose-6-phosphate dehydrogenase ZWF1 6-phosphogluconide lactonease encoding gene PGL and the gene encoding 6-phosphoglucate dehydrogenase GND1 The expression cassette was integrated into a plasmid and linearized, then transformed into a second recombinant Yersinia lipophila strain to obtain the final product.

3. The use of the engineered strain of claim 1 that produces 2'-fucosylated lactose in the production of 2'-fucosylated lactose.

4. The application of the engineered strain producing 2'-fucosylated lactose as described in claim 3 in the production of 2'-fucosylated lactose, characterized in that, The engineered strain that produces 2'-fucosylated lactose was activated and inoculated into a seed culture medium. The resulting seed culture was then inoculated into a fermentation medium, and 2'-fucosylated lactose was obtained through fermentation.

5. The application of the engineered strain producing 2'-fucosylated lactose as described in claim 4 in the production of 2'-fucosylated lactose, characterized in that, The seed culture inoculation amount is 1~10% (V / V).

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