A genetically engineered bacterium for producing 2'-fucosyllactose based on mannitol, a construction method and application thereof

Abstract

The present application belongs to the field of microorganism and fermentation engineering technology, and particularly relates to a genetically engineered bacterium for producing 2'-fucosyllactose based on mannitol and a construction method and application thereof. Specifically, on the basis of the previously constructed 2'-fucosyllactose-producing engineered strain, the 2'-fucosyllactose synthesis pathway is iteratively integrated, the GDP-D-mannose synthesis pathway is enhanced, the hexose transporter Yht1 is overexpressed, the GDP-D-mannose synthesis pathway is further enhanced, and the hexose transporter Yht3 is knocked out, so that the Yarrowia lipolytica strain can efficiently absorb mannitol for growth metabolism and synthesis of 2'-fucosyllactose. The present application is the first report of using mannitol as a substrate for the production of 2'-fucosyllactose, and has important industrial application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms and fermentation engineering, and particularly relates to a genetically engineered bacterium for producing 2'-fucosyllactose based on mannitol as well as a construction method and application thereof. BACKGROUND

[0002] The information disclosed in the background of the present application is only intended to increase the understanding of the overall background of the present application, and should not necessarily be regarded as acknowledging or implicitly suggesting that this information constitutes prior art known to those of ordinary skill in the art.

[0003] The biosynthesis of 2'-fucosyllactose (2'-FL) using Yarrowia lipolytica has been proven to have great application value. However, the carbon source used in the fermentation production of 2'-fucosyllactose using Yarrowia lipolytica is still the traditional carbon source glucose. In large-scale industrial production, the use of glucose has problems such as land cultivation and grain competition, low utilization rate, etc., which does not meet the current social requirements for rational use of resources, and the conversion rate of carbon source is low when using glucose as the substrate for fermentation, which has the problems of environmental protection and low utilization rate in industrial application.

[0004] With the continuous research on the development and utilization of non-grain biomass resources, algal biomass resources have gradually entered people's field of vision due to their characteristics of not occupying agricultural land resources, high photosynthetic efficiency, and easy cultivation. Mannitol is one of the most abundant carbon-containing compounds in brown algae, and its extraction method is simple, which can be obtained by hot water soaking only. In addition, mannitol has higher reducing power and stimulates the metabolic process of microorganisms. Therefore, microbial fermentation with mannitol as the carbon source has the characteristics of low cost and high yield, and is the best raw material for large-scale industrial microbial biosynthesis. However, the inventors found that there is no report on the biosynthesis of 2'-fucosyllactose using mannitol as the substrate in the prior art. SUMMARY

[0005] To address the problems existing in the prior art, the present invention aims to provide a genetically engineered strain for the production of 2′-fucosylated lactose based on mannitol, its construction method, and its applications. Specifically, the present invention enables *Yarrowia lipolytica* to produce 2′-fucosylated lactose through metabolic engineering. The expression of transport proteins is optimized to enhance the absorption and utilization of mannitol, and the synthesis of GDP-D-mannose is enhanced by overexpressing key enzyme genes in the GDP-D-mannose synthesis pathway. The resulting recombinant *Yarrowia lipolytica* strain possesses the ability to efficiently utilize mannitol to produce 2′-fucosylated lactose. Based on the above research results, the present invention is thus completed.

[0006] Specifically, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a genetically engineered bacterium for the production of 2′-fucosylated lactose based on mannitol. The genetically engineered bacterium is obtained by overexpressing the encoding genes of hexose transporter Yht1, phosphogmannose mutase and 1-phosphogmannose guanylate transferase, and knocking out the encoding gene of hexose transporter Yht3.

[0008] The *Yersinia lipolytica* strain with 2′-fucosylated lactose production capacity can be a process strain from the engineered strain in CN116622534A that produces high levels of 2′-fucosylated lactose. This process strain can express lactose transporter, GDP-mannose dehydratase, GDP-fucosylated synthase, and α-1,2-fucosylated transferase fused with a Ub tag. It can also 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. This strain can be the second recombinant *Yersinia lipolytica* strain in CN116622534A (or the F4-2 strain in the examples thereof).

[0009] In a second aspect, the present invention provides a method for constructing the above-mentioned genetically engineered bacteria, the method comprising: constructing and linearizing an integrative plasmid containing the expression cassette of the hexose transporter Yht1 gene YHT1; and integrating the linearized plasmid into the genome of Yersinia lipolytica with the ability to produce 2′-fucosylated lactose by an iterative integration method to obtain a first Yersinia lipolytica strain of engineered bacteria.

[0010] An integrated plasmid containing the expression cassette encoding the phosphogluconomutase gene SEC53 and the 1-phosphogluconoguanylate transferase gene PSA1 was constructed and linearized, and then transformed into the first engineered strain of Yersinia lipolytica to obtain the second engineered strain of Yersinia lipolytica.

[0011] A free plasmid for knocking out the hexose transporter Yht3 gene YHT3 was constructed and transformed into the second lipophilic Yersinia strain.

[0012] A third aspect of the present invention provides the application of the above-mentioned genetically engineered bacteria in the production of 2′-fucosylated lactose. Specifically, the application is: the production of 2′-fucosylated lactose using mannitol as a carbon source.

[0013] A fourth aspect of the present invention provides a method for producing 2′-fucosylated lactose based on mannitol, the method comprising: adding the genetically engineered bacteria to a culture medium containing mannitol for fermentation culture, and separating and purifying 2′-fucosylated lactose.

[0014] The mannitol-containing culture medium components include: 6-8% mannitol; 2-4% tryptone; 1-2% yeast extract; and 0.4-1% lactose.

[0015] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0016] Based on the previously constructed engineered strain producing 2′-fucosylvate, the above technical solution iteratively integrates the 2′-fucosylvate synthesis pathway, enhances the GDP-D-mannose synthesis pathway, overexpresses the hexose transporter Yht1, further enhances the GDP-D-mannose synthesis pathway, and knocks out the hexose transporter Yht3, enabling the *Yarrowia lipolytica* strain to efficiently absorb mannitol for growth and metabolism and synthesize 2′-fucosylvate. After fermentation, the strain achieved a yield of 9.03 g / L in shake-flask fermentation medium, higher than the yield when using glucose as a carbon source. In a 5L fermenter using rich and inexpensive media, the 2′-fucosylvate yield reached 30.05 g / L and 27.55 g / L, respectively. This is the first report of 2′-fucosylvate production using mannitol as a substrate, and has significant industrial application value. Attached Figure Description

[0017] 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.

[0018] Figure 1 This is a graph showing the yield of 2′-FL from eight transformants of the F1 series strain in this invention.

[0019] Figure 2 This is a graph showing the yield of 2′-FL from eight transformants of the F2 series strains in this invention.

[0020] Figure 3 This is a graph showing the yield of 2′-FL from eight transformants of the F3 series strain in this invention.

[0021] Figure 4 This is a graph showing the yield of 2′-FL from eight transformants of the F4 series strain in this invention.

[0022] Figure 5 This is a graph showing the 2′-FL yield of the process strain F4-2 in YPD and YPM media during fermentation in this invention.

[0023] Figure 6 This is a graph showing the 2′-FL yield of strains MF1, MF2, and MF3 in the process of this invention.

[0024] Figure 7 This is a graph showing the yield of 2′-FL produced by the engineered strain MF3 using YPM medium in a fermenter during the fermentation process of this invention.

[0025] Figure 8 This is a graph showing the yield of 2′-FL produced by the engineered strain MF3 in this invention using CM1 medium in a fermentation tank. Detailed Implementation

[0026] It should be noted that the following detailed description is illustrative and intended to provide further explanation 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.

[0027] 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.

[0028] Given that the current carbon source used by Yersinia lipolytica to produce 2′-fucosylated lactose is a traditional carbon source with low glucose yield, this invention proposes an engineered strain that produces high 2′-fucosylated lactose, its construction method, and its application.

[0029] In a typical embodiment of the present invention, a genetically engineered bacterium for the production of 2′-fucosylated lactose based on mannitol is provided. The genetically engineered bacterium is obtained by overexpressing the encoding genes of hexose transporter Yht1, phosphogmannose mutase and 1-phosphogmannose guanylate transferase, and knocking out the encoding gene of hexose transporter Yht3.

[0030] The *Yersinia lipolytica* strain with 2′-fucosylated lactose production capacity can be a process strain from the engineered strain in CN116622534A that produces high levels of 2′-fucosylated lactose. This process strain can express lactose transporter, GDP-mannose dehydratase, GDP-fucosylated synthase, and α-1,2-fucosylated transferase fused with a Ub tag. It can also 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. This strain can be the second recombinant *Yersinia lipolytica* strain in CN116622534A (or the F4-2 strain in the examples thereof).

[0031] In this invention, overexpression of the hexose transporter Yht1 enhances mannitol transport, overexpression of phospmannose mutase and 1-phospmannose guanylate transferase further enhances the GDP-D-mannose synthesis pathway, and knockout of the hexose transporter Yht3 promotes mannitol uptake. Using the above construction method, a recombinant Yersinia lipolytica strain with high efficiency in producing 2′-fucosylated lactose from mannitol was finally obtained.

[0032] In this invention, the gene encoding the hexose transporter Yht1 is the YHT1 gene, with the gene number YALI1_C08523g;

[0033] The gene encoding the phosphogluconomutase is the SEC53 gene, with the gene number YALI0_D13112g.

[0034] The gene encoding the 1-phosphate mannose-guanosine transferase is the PSA1 gene, with the gene number YALI0_C06490g.

[0035] The gene encoding the hexose transporter Yht3 is the YHT3 gene, with the gene number YALI1_F25587g.

[0036] In another specific embodiment of the present invention, a method for constructing the above-mentioned genetically engineered bacteria is provided. The method includes: constructing and linearizing an integrative plasmid containing the expression cassette of the hexose transporter Yht1 gene YHT1; and integrating the linearized plasmid into the genome of Yersinia lipolytica with the ability to produce 2′-fucosylated lactose by an iterative integration method to obtain a first Yersinia lipolytica strain engineered bacteria.

[0037] An integrated plasmid containing the expression cassette encoding the phosphogluconomutase gene SEC53 and the 1-phosphogluconoguanylate transferase gene PSA1 was constructed and linearized, and then transformed into the first engineered strain of Yersinia lipolytica to obtain the second engineered strain of Yersinia lipolytica.

[0038] A free plasmid for knocking out the hexose transporter Yht3 gene YHT3 was constructed and transformed into the second lipophilic Yersinia strain.

[0039] The *Yersinia lipolytica* strain with the ability to produce 2′-fucosylated lactose is the second recombinant *Yersinia lipolytica* strain in CN116622534A (or the F4-2 strain in the examples thereof).

[0040] In another specific embodiment of the present invention, the construction method includes:

[0041] The linearized pTrp-YHT1 plasmid was transformed into *Yersinia lipolytica* strain with 2′-fucosylated lactose production capacity by knocking out the Trp gene, thus obtaining the first engineered *Yersinia lipolytica* strain; wherein, the pTrp-YHT1 plasmid is an integrative plasmid containing the expression cassette of the gene encoding the hexose transporter Yht1, YHT1.

[0042] In another specific embodiment of the present invention, the YHT1 gene and its gene-regulated promoter and terminator are used to construct the YHT1 gene expression cassette via fusion PCR, and the YHT1 gene expression cassette is ligated to a vector backbone containing the Trp gene expression cassette via Gibson assembly to obtain the pTrp-YHT1 plasmid.

[0043] The linearized pTrp-SP plasmid was transformed into the first engineered strain of *Yersinia lipolytica* with the Trp gene knocked out to obtain the second engineered strain of *Yersinia lipolytica*. The pTrp-SP plasmid was an integrated plasmid containing the expression cassettes of the phosphogmannose mutase encoding gene SEC53 and the 1-phosphogmannose guanylate transferase encoding gene PSA1.

[0044] In another specific embodiment of the present invention, the SEC53 gene, the PSA1 gene, and their respective gene-regulated promoters and terminators are constructed by fusion PCR to form the SEC53 gene expression cassette and the PSA1 gene expression cassette. The SEC53 gene expression cassette and the PSA1 gene expression cassette are then linked to the vector backbone of the Trp gene expression cassette by Gibson assembly to obtain the pTrp-SP plasmid.

[0045] The above-mentioned genetically engineered bacteria were obtained by transforming pCas9-YHT3 into the second strain of Yersinia lipophila; wherein, the pCas9-YHT3 plasmid is a free knockout plasmid with the YHT3 gene knockout site sequence.

[0046] In another specific embodiment of the present invention, the YHT3 gene knockout site sequence is constructed into a plasmid containing Cas9 protein by primer synthesis to obtain the pCas9-YHT3 plasmid.

[0047] In another specific embodiment of the present invention, the application of the above-mentioned genetically engineered bacteria in the production of 2′-fucosylated lactose is provided. Specifically, the application is: producing 2′-fucosylated lactose using mannitol as a carbon source.

[0048] In another specific embodiment of the present invention, a method for producing 2′-fucosylated lactose based on mannitol is provided, the method comprising: adding the genetically engineered bacteria to a culture medium containing mannitol for fermentation culture, and separating and purifying to obtain 2′-fucosylated lactose.

[0049] The mannitol-containing culture medium components include: 6-8% mannitol; 2-4% tryptone; 1-2% yeast extract; and 0.4-1% lactose.

[0050] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0051] Example

[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, PO1f (ATCC number MYA-2613; genotype MATAura3-3021leu2-270xpr2-322axp2-deltaNU49XPR2::SUC2), was purchased from ATCC.

[0057] 5. The genes involved in this embodiment, namely GDP-mannose dehydratase (GMD) from *Morchella alpina*, lactose transporter (LAC12) from *Kluyveromyces lactis*, and GDP-fucose synthase (GMER) and fucosyltransferase (FutC) from *Helicobacter pylori*, were all obtained through gene synthesis.

[0058] 6. LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder.

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

[0060] YPD medium: 1% yeast extract, 2% tryptone, 2% glucose.

[0061] YPD solid medium: 1% yeast extract, 2% tryptone, 2% glucose, 2% agar powder.

[0062] YPM medium: 1% yeast extract, 2% tryptone, 2% mannitol.

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

[0064] CM1 culture medium: 0.5 g / L magnesium sulfate heptahydrate, 2.3 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate; Vitamin stock solution formula: 0.05 g / L biotin, 1 g / L vitamin B5, 1 g / L vitamin B3, 25 g / L inositol, 1 g / L thiamine hydrochloride, 1 g / L pyridoxine hydrochloride, 0.2 g / L para-aminobenzoic acid; Trace element stock solution formula: 15 g / L Disodium EDTA, 4.5 g / L zinc sulfate heptahydrate, 0.84 g / L manganese chloride dihydrate, 0.3 g / L cobalt chloride hexahydrate, 0.3 g / L copper sulfate pentahydrate, 0.4 g / L sodium molybdate dihydrate, 4.5 g / L calcium chloride dihydrate, 3 g / L ferric sulfate heptahydrate, 1 g / L boric acid, 0.1 g / L potassium iodide; when using, add 50 μl of vitamin storage solution and 50 μl of trace element storage solution to 50 mL of culture medium respectively.

[0065] II. Preparation of the target gene:

[0066] 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 hpGMER.

[0067] 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.

[0068] 4. Endogenous genes including PMI40, SEC53 and PSA1 were obtained by PCR amplification using the genome of strain PO1f as a template.

[0069] 5. The Ub-futC sequence is as follows:

[0070] >SEQ ID NO.1

[0071]

[0072] Table 1 Primer sequences and knockout site sequences

[0073]

[0074]

[0075] III. Plasmid Construction:

[0076] 1. Construction of plasmids pLEP-futC-GGL and pLEP-Ub-futC-GGL

[0077] The plasmid pLEP-futC-GGL was constructed primarily via fusion PCR, amplifying the klLAC12, maGMD, and hpGMER 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 hpGMER genes were cloned into the pLEP-futC plasmid via Gibson assembly.

[0078] The construction method of plasmid pLEP-Ub-futC-GGL involves first amplifying the optimized Ub tag and futC gene separately by PCR, and then fusing the two genes using fusion PCR. The resulting fusion gene is then cloned into the pLEP-leu plasmid using Gibson assembly to obtain the pLEP-Ub-futC plasmid. Similarly, the klLAC12, maGMD, and hpGMER gene expression cassettes are cloned into the pLEP-Ub-futC plasmid.

[0079] 2. Construction of plasmid pULUF-GG

[0080] 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 hpGMER genes, along with the promoters and terminators for the regulation of 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.

[0081] 3. Construction of plasmid pHLUF-GG

[0082] 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.

[0083] 4. Construction of plasmid pMAN

[0084] 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. 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.

[0085] 5. Construction of plasmid pTrp-YHT1

[0086] 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. Using the genome of PO1f strain as a template, the YHT1 gene was amplified and constructed at the Bsp119I restriction site of the plasmid to construct the expression plasmid pTrp-YHT1.

[0087] 6. Construction of plasmid pCas9-YHT3

[0088] The pCas9-YHT3 plasmid was obtained by constructing a 20bp sequence near the selected YHT3 gene knockout site into a plasmid containing the Cas9 gene using gene synthesis.

[0089] Example 2: Construction of an engineered Yarrowia lipolytica strain that produces high levels of 2′-fucosylated lactose.

[0090] 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, thereby 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 cells of yeast lithium acetate. (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 water bath at 30℃ 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℃, 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 yield of 2′-FL in the pLEP-futC-GGL transformed strain was 0.10 g / L, while the yield of 2′-FL 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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 Yersinia 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 subsequently 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.

[0095] 6. The obtained F4-2 strain was subjected to shake-flask fermentation experiments in YPD liquid medium and YPM liquid medium, respectively. After 144 h, 2′-FL was detected. The detection method for 2′-fucosylated lactose was the same as described above. The results showed that the yield of 2′-fucosylated lactose in YPM liquid medium was 4.09 g / L, approximately twice that in YPD liquid medium. Figure 5 As shown in the figure. Comparing the cell productivity under the two culture conditions, it was found that the 2′-fucosylated lactose titer of cells in YPM was 0.166, which was 62.7% higher than that in YPD. The 2′-fucosylated lactose yield under mannitol treatment (0.106 g / g) was significantly higher than that under glucose treatment (0.027 g / g), indicating that mannitol has a more efficient 2′-fucosylated lactose yield.

[0096] 7. Using recombinant Yersinia lipolyticis F4-2 as the starting strain, the Trp gene was first knocked out of the genome using CRISPR / Cas9. Then, the linearized plasmid pTrp-YHT1 was transformed into the resulting Trp gene knockout strain. Transformants were selected and fermented in YPM liquid medium for verification. The results showed that the OD of transformant MF1 was... 600 And the yield of 2′-FL was significantly higher than that of strain F4-2, such as Figure 6 As shown, overexpression of YHT1 can effectively promote the absorption and utilization of mannitol by Yersinia lipophila strain and further promote the accumulation of 2′-FL.

[0097] 8. To further promote intracellular GDP-D-mannose production, the linearized pTrp-SP plasmid was transformed into strain MF1 to enhance the GDP-D-mannose synthesis pathway. The specific transformation method is as described above. Transformants were picked and fermented in YPM liquid medium. After 144 hours, 2′-FL was detected. The detection method for 2′-fucosylated lactose was as described above. The results showed that strain MF2 had the highest 2′-FL yield, reaching 7.68 g / L. Figure 6 As shown.

[0098] 9. In *Yarrowia lipolytica* cells, there are some transport proteins that may inhibit mannitol absorption. Therefore, the gene encoding the hexose transporter yht3, which may inhibit mannitol absorption, was knocked out. Using recombinant *Yarrowia lipolytica* strain MF2 as the starting strain, the YHT3 gene in the genome was knocked out using the CRISPR / Cas9 method. Transformants were selected and fermented in YPM liquid medium for verification. The results showed that the 2′-fucosylated lactose production of transformant MF3 was significantly increased, reaching 9.03 g / L. Figure 6 As shown.

[0099] Example 3: Continuous fed fermentation culture of strain MF3 in YPM medium

[0100] The recombinant strain MF3, capable of high production of 2′-FL constructed in Example 2, was subjected to a fed-batch fermentation experiment in a 5L fermenter. The culture medium used in this example was YPM medium (2% tryptone, 1% yeast extract, 6% mannitol, 0.7% lactose), with an initial volume of 3.6L. The bacterial culture was taken from the glycerol inoculum 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 1.5 v / v / m, and a stirring speed of 750 rpm. As the dissolved oxygen concentration increased, the stirring speed was reduced to 500 rpm, the aeration rate was reduced to 1 v / v / m, and the pH was maintained at 5.5. Mannitol concentration in the fermenter was sampled every 6 hours. Once the initial mannitol in the culture medium was depleted, mannitol was replenished in batches. Biomass, lactose consumption, and 2′-FL yield in the fermenter were sampled every 6 hours. Figure 6 The fermentation results showed that the yield of 2′-FL reached its highest level of 30.09 g / L after 144 hours of fermentation. Figure 7 As shown.

[0101] Example 4: Continuous fed-batch fermentation culture of strain MF3 in CM1 medium

[0102] Considering the high cost of YPM medium, the recombinant strain MF3, which produced high levels of 2′-FL as described in Example 2, was used in a 5L fermenter for fed-batch fermentation using inexpensive inorganic salt medium. The medium used in this example was CM1 medium (specific components as described above: 7% mannitol, 0.7% lactose), with an initial volume of 3.6L. The bacterial culture was taken from the glycerol culture tube and streaked onto a YPD agar plate for 24 hours. The resulting single colonies were inoculated into a shake flask containing 50 mL of YPD medium and incubated at 30°C and 220 rpm for 24 hours. This seed culture was then transferred to a shake flask containing 200 mL of YPD medium and incubated for another 24 hours. Finally, this seed culture was inoculated into the 5L fermenter. The fermenter was set to a temperature of 30℃, an aeration rate of 1.5 v / v / m, and a stirring speed of 750 rpm. As the dissolved oxygen concentration increased, the stirring speed was reduced to 500 rpm, and the aeration rate was reduced to 1 v / v / m. The pH was maintained at 5.5. Mannitol concentration in the fermenter was sampled every 6 hours. Once the initial mannitol in the culture medium was depleted, mannitol was added in batches. Biomass, lactose consumption, and 2′-FL production were sampled every 6 hours. Figure 6 The fermentation results showed that the yield of 2′-FL reached its highest level of 27.55 g / L after 158 hours of fermentation. Figure 8 As shown.

[0103] Matters not covered in this invention are common knowledge.

[0104] 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. A genetically engineered bacterium, characterized in that, Overexpression of Yersinia lipophila in the starting strain ( Yarrowia lipolytica The genes encoding hexose transporter Yht1, phosphogmannose mutase SEC53, and 1-phosphogmannose guanylate transferase PSA1 derived from PO1f were obtained by knocking out the gene encoding hexose transporter Yht3. The method for constructing the starting strain includes the following steps: The construction contains Kluyveromyces lactis ( Kluyveromyces lactis lactose transporter gene klLAC12 Originating from alpine spores ( Mortierella alpina GDP-mannose dehydrase gene maGMD Originating from Helicobacter pylori ( Helicobacter pylori GDP-fucose synthase gene hpGMER And as shown in SEQ ID NO.1 Ub-futC The expression cassette was integrated into a linearized plasmid and then integrated into the genome of Yersinia lipophila PO1f using an iterative integration method to obtain the first recombinant Yersinia lipophila strain. Construct a gene encoding mannose-6-phosphate isomerase derived from Yersinia lipolytica. 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, then transformed into the first recombinant Yersinia lipophila strain to obtain the starting strain. The preservation number of the *Yersinia lipophila* PO1f is ATCC MYA-2613.

2. The method for constructing the genetically engineered bacteria according to claim 1, characterized in that, The construction method includes: constructing a gene-containing... YHT1 The expression cassette was integrated into a plasmid and linearized. The linearized plasmid was then integrated into the genome of the starting strain using an iterative integration method to obtain the first engineered Yeast lipophilia strain. Build includes SEC53 and PSA1 The expression cassette was integrated into a plasmid and linearized, and then transformed into the first engineered strain of Yersinia lipophila to obtain the second engineered strain of Yersinia lipophila. Constructing a gene knockout YHT3 The free plasmid was transformed into the engineered strain of Yersinia lipophila to obtain the product.

3. The construction method as described in claim 2, characterized in that, The construction method includes: The linearized pTrp-YHT1 plasmid was transformed into the knockout plasmid. Trp The first engineered *Yarrowia lipolytica* strain was obtained from the starting strain of the gene; among them, the pTrp-YHT1 plasmid contains the gene encoding the hexose transporter Yht1. YHT1 Integral plasmids containing expression frames; The linearized pTrp-SP plasmid was transformed into the first engineered *Yersinia lipolytica* strain to obtain the second engineered *Yersinia lipolytica* strain; wherein the pTrp-SP plasmid contains the gene encoding phosphoglucono-mannose mutase. SEC53 and the gene encoding 1-phosphoguanosine monophosphate transferase PSA1 Integral plasmids containing expression frames; The genetically engineered strain was obtained by transforming pCas9-YHT3 into a second strain of *Yersinia lipophila*; wherein the pCas9-YHT3 plasmid was a vector containing pCas9-YHT3. YHT3 Free knockout plasmids containing gene knockout site sequences.

4. The application of the genetically engineered bacteria according to claim 1 in the production of 2'-fucosylated lactose, characterized in that, The specific application is the production of 2'-fucosylated lactose using mannitol as a carbon source.

5. A method for producing 2'-fucosylated lactose based on mannitol, characterized in that, The method includes: adding the genetically engineered bacteria of claim 1 to a culture medium containing mannitol for fermentation culture, and separating and purifying 2'-fucosylated lactose.

6. The method as described in claim 5, characterized in that, The mannitol-containing culture medium components include: mannitol 6-8%; tryptone 2-4%; yeast extract 1-2%; and lactose 0.4-1%.

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