Genetically engineered bacterium for producing 2 '-fucosyllactose based on mannitol as well as construction method and application of genetically engineered bacterium

Abstract

The invention 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 of the genetically engineered bacterium. Specifically, on the basis of an engineering strain for producing 2 '-fucosyllactose, which is constructed in an earlier stage, 2'-fucosyllactose is produced; through iterative integration of a 2 '-fucosyllactose synthesis pathway, enhancement of a GDP-D-mannose synthesis pathway, overexpression of hexose transporter protein Yht1, further enhancement of the GDP-D-mannose synthesis pathway and knockout of hexose transporter protein Yht3, the Yarrowia lipolytica strain can efficiently absorb mannitol for growth metabolism and synthesize the 2'-fucosyllactose. According to the invention, the mannitol is firstly used as a substrate for production of the 2 '-fucosyllactose, and the mannitol has important industrial application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and fermentation engineering, and specifically relates to a genetic engineering bacterium for producing 2′-fucosyllactose based on mannitol, and a construction method and application thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance the understanding of the overall background of the invention and should not be necessarily regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] The biosynthesis of 2′-fucosyllactose (2′-FL) using Yarrowia lipolytica has been shown 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 raw material cultivation competing with grain and low utilization rate, which does not meet the requirements of today's society for rational resource utilization. In addition, when glucose is used as a substrate for fermentation, the conversion rate of the carbon source is low, and there are problems such as environmental protection and low utilization rate in industrial applications.

[0004] With the continuous research on the development and utilization of non-food biomass resources, algae biomass resources have gradually come into people's view due to the characteristics of not occupying agricultural arable land resources, high photosynthetic efficiency, and easy cultivation. Mannitol is one of the most abundant carbon-containing compounds in brown algae. Its extraction method is simple and can be obtained by soaking in hot water. In addition, mannitol has a higher reducing ability and stimulates the metabolic process of microorganisms. Therefore, microbial fermentation with mannitol as a 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 a substrate in the prior art. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide a genetically engineered bacterium for producing 2′-fucosyllactose based on mannitol, and its construction method and application. Specifically, the present invention enables Yarrowia lipolytica to produce 2′-fucosyllactose through metabolic engineering, enhances the ability to absorb and utilize mannitol by optimizing the expression of transport proteins, and enhances the synthesis of GDP-D-mannose by overexpressing key enzyme genes in the GDP-D-mannose synthesis pathway. The obtained recombinant Yarrowia lipolytica strain has the ability to efficiently utilize mannitol to produce 2′-fucosyllactose. Based on the above research results, the present invention is completed.

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

[0007] In a first aspect of the present invention, a genetically engineered bacterium is provided for producing 2′-fucosyllactose based on mannitol. The genetically engineered bacterium is obtained by using Yarrowia lipolytica having the ability to produce 2′-fucosyllactose as a starting strain, overexpressing the coding genes of the hexose transporter Yht1, phosphomannose mutase and 1-phosphomannose guanosyltransferase, and knocking out the coding gene of the hexose transporter Yht3;

[0008] Among them, the Yarrowia lipolytica having the ability to produce 2′-fucosyllactose can be a process strain in the engineered strain for constructing a high-yield 2′-fucosyllactose in CN116622534A, and the process strain can express lactose transporter, GDP-mannose dehydratase, GDP-fucose synthase and α-1,2-fucosyltransferase fused with a Ub tag, and can enhance the GDP-D-mannose synthesis pathway by overexpressing 6-phosphomannose isomerase, phosphomannose mutase and 1-phosphomannose guanyl transferase using a strong promoter, that is, it can be the second recombinant Yarrowia lipolytica strain in CN116622534A (or the F4-2 strain in its embodiment).

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

[0010] An integrative plasmid containing expression frames of the phosphomannose mutase encoding gene SEC53 and the 1-phosphomannose guanosyltransferase encoding gene PSA1 is constructed and linearized, and transformed into the first Yarrowia lipolytica engineered bacteria to obtain a second Yarrowia lipolytica engineered bacteria;

[0011] An episomal plasmid for knocking out the hexose transporter Yht3 gene YHT3 was constructed and transformed into the second Yarrowia lipolytica engineered bacteria to obtain the result.

[0012] The third aspect of the present invention provides the use of the above genetically engineered bacteria in the production of 2′-fucosyllactose. Specifically, the use is: producing 2′-fucosyllactose using mannitol as a carbon source.

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

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

[0015] Beneficial technical effects of one or more of the above technical solutions:

[0016] The above technical scheme is based on the engineering strain producing 2′-fucosyllactose constructed in the early stage, and iteratively integrates the 2′-fucosyllactose 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, so that the Yarrowia lipolytica strain can efficiently absorb mannitol for growth metabolism and synthesize 2′-fucosyllactose. After cultivation and fermentation, the yield of the strain in the shake flask fermentation medium reaches 9.03g / L, which is higher than the shake flask fermentation yield when glucose is used as the carbon source; in a 5L fermenter, rich medium and cheap medium are used for fermentation and culture, and the 2′-fucosyllactose yield reaches 30.05g / L and 27.55g / L respectively. This is the first report on the production of 2′-fucosyllactose using mannitol as a substrate, which has important industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0018] Figure 1 This is a graph showing the 2′-FL production of 8 transformants of the process strain F1 series of bacteria in the present invention.

[0019] Figure 2 This is a graph showing the 2′-FL production of 8 transformants of the process strain F2 series of bacteria in the present invention.

[0020] Figure 3 This is a graph showing the 2′-FL production of 8 transformants of the process strain F3 series of bacteria in the present invention.

[0021] Figure 4 This is a graph showing the 2′-FL production of 8 transformants of the process strain F4 series of bacteria in the present invention.

[0022] Figure 5 This is a graph showing the 2′-FL production of the process strain F4-2 in the present invention when fermented in YPD medium and YPM medium.

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

[0024] Figure 7 This is a graph showing the yield of 2′-FL produced by fermentation of the engineered bacteria MF3 using YPM medium in a tank.

[0025] Figure 8 This is a graph showing the yield of 2′-FL produced by fermentation of the engineered bacteria MF3 using CM1 medium in a tank. DETAILED DESCRIPTION

[0026] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0027] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0028] Considering that the carbon source currently used by Yarrowia lipolytica to produce 2′-fucosyllactose is a traditional carbon source with low glucose yield, the present invention provides an engineered strain with high 2′-fucosyllactose yield, and a construction method and application thereof.

[0029] A typical embodiment of the present invention provides a genetically engineered bacterium for producing 2′-fucosyllactose based on mannitol, wherein the genetically engineered bacterium is obtained by using Yarrowia lipolytica having the ability to produce 2′-fucosyllactose as a starting strain, overexpressing the coding genes of the hexose transporter Yht1, phosphomannose mutase and 1-phosphomannose guanosyltransferase, and knocking out the coding gene of the hexose transporter Yht3;

[0030] Among them, the Yarrowia lipolytica having the ability to produce 2′-fucosyllactose can be a process strain in the engineered strain for constructing a high-yield 2′-fucosyllactose in CN116622534A, and the process strain can express lactose transporter, GDP-mannose dehydratase, GDP-fucose synthase and α-1,2-fucosyltransferase fused with a Ub tag, and can enhance the GDP-D-mannose synthesis pathway by overexpressing 6-phosphomannose isomerase, phosphomannose mutase and 1-phosphomannose guanyl transferase using a strong promoter, that is, it can be the second recombinant Yarrowia lipolytica strain in CN116622534A (or the F4-2 strain in its embodiment).

[0031] In the present invention, overexpression of the hexose transporter Yht1 is beneficial to enhancing the transport of mannitol, overexpression of phosphomannose mutase and 1-phosphomannose guanylyltransferase is beneficial to further enhancing the GDP-D-mannose synthesis pathway, and knocking out the hexose transporter Yht3 promotes the absorption of mannitol. The above construction method is used to finally obtain a recombinant Yarrowia lipolytica strain capable of efficiently utilizing mannitol to produce 2′-fucosyllactose.

[0032] In the present invention, the coding gene of the hexose transporter Yht1 is the YHT1 gene, and the gene number is YALI1_C08523g;

[0033] The coding gene of the phosphomannose mutase is the SEC53 gene, and the gene number is YALI0_D13112g;

[0034] The coding gene of 1-phosphomannose guanosyltransferase is PSA1 gene, and the gene number is YALI0_C06490g;

[0035] The coding gene of the hexose transporter Yht3 is the YHT3 gene, and the gene number is 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 comprising: constructing an integrative plasmid containing an expression frame of the hexose transporter Yht1 gene YHT1 and linearizing it, integrating the linearized plasmid into the genome of Yarrowia lipolytica having the ability to produce 2′-fucosyllactose by an iterative integration method, to obtain a first Yarrowia lipolytica strain engineered bacteria;

[0037] An integrative plasmid containing expression frames of the phosphomannose mutase encoding gene SEC53 and the 1-phosphomannose guanosyltransferase encoding gene PSA1 is constructed and linearized, and transformed into the first Yarrowia lipolytica engineered bacteria to obtain a second Yarrowia lipolytica engineered bacteria;

[0038] An episomal plasmid for knocking out the hexose transporter Yht3 gene YHT3 was constructed and transformed into the second Yarrowia lipolytica engineered bacteria to obtain the result.

[0039] Wherein, the Yarrowia lipolytica having the ability to produce 2′-fucosyllactose is the second recombinant Yarrowia lipolytica strain in CN116622534A (or the F4-2 strain in its examples).

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

[0041] The linearized pTrp-YHT1 plasmid was transformed into Yarrowia lipolytica with Trp gene knocked out and capable of producing 2′-fucosyllactose to obtain the first Yarrowia lipolytica strain engineered bacteria; wherein the pTrp-YHT1 plasmid is an integrative plasmid containing the expression frame of the gene YHT1 encoding the hexose transporter Yht1.

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

[0043] The linearized pTrp-SP plasmid is transformed into a first Yarrowia lipolytica strain engineered bacterium in which the Trp gene is knocked out to obtain a second Yarrowia lipolytica strain engineered bacterium; wherein the pTrp-SP plasmid is an integrative plasmid containing expression frames of the phosphomannose mutase encoding gene SEC53 and the 1-phosphomannose guanosyltransferase 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 respectively used to construct the SEC53 gene expression cassette and the PSA1 gene expression cassette by fusion PCR, and the SEC53 gene expression cassette and the PSA1 gene expression cassette are connected to the vector backbone marked with the Trp gene expression cassette by Gibson assembly to obtain the pTrp-SP plasmid.

[0045] pCas9-YHT3 was transformed into a second Yarrowia lipolytica strain engineered bacteria to obtain the above-mentioned genetically engineered bacteria; wherein the pCas9-YHT3 plasmid is a free knockout plasmid with a YHT3 gene knockout site sequence.

[0046] In another specific embodiment of the present invention, the YHT3 gene knockout site sequence is constructed onto a plasmid containing the 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 genetically engineered bacteria in the production of 2′-fucosyllactose is provided. Specifically, the application is to produce 2′-fucosyllactose using mannitol as a carbon source.

[0048] In another specific embodiment of the present invention, a method for producing 2′-fucosyllactose 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′-fucosyllactose.

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

[0050] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0051] Example

[0052] 1. Materials and Methods

[0053] 1. The gene synthesis, primer synthesis and sequencing in this example were completed by Beijing Qingke Xinye Biotechnology Co., Ltd.

[0054] 2. The experimental methods used in this example, including plasmid construction, enzyme digestion, preparation of competent cells, transformation, etc., are all conventional methods unless otherwise specified. If necessary, the specific experimental conditions can be determined by simple experiments.

[0055] 3. Unless otherwise specified, the materials and reagents used in this example can be obtained from commercial sources.

[0056] 4. The original strain of Yarrowia lipolytica PO1f (ATCC No. MYA-2613; genotype MATAura3-3021leu2-270xpr2-322axp2-deltaNU49XPR2::SUC2) involved in this example was purchased from ATCC.

[0057] 5. The genes involved in this example, GDP-mannose dehydratase (GMD) from Mortierella alpina, lactose transporter (LAC12) from Kluyveromyces lactis, GDP-fucose synthase (GMER) and fucosyltransferase (FutC) encoding genes from Helicobacter pylori were all obtained by 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: 0.5g / L magnesium sulfate heptahydrate, 2.3g / L ammonium sulfate, 3g / L potassium dihydrogen phosphate; vitamin storage solution formula is as follows: 0.05g / L biotin, 1g / L vitamin B5, 1g / L vitamin B3, 25g / L inositol, 1g / L thiamine hydrochloride, 1g / L pyridoxine hydrochloride, 0.2g / L p-aminobenzoic acid; trace element storage solution formula is as follows: 15g / L Disodium EDTA, 4.5g / L zinc sulfate heptahydrate, 0.84g / L manganese chloride dihydrate, 0.3g / L cobalt chloride hexahydrate, 0.3g / L copper sulfate pentahydrate, 0.4g / L sodium molybdate dihydrate, 4.5g / L calcium chloride dihydrate, 3g / L ferric sulfate heptahydrate, 1g / L boric acid, 0.1g / L potassium iodide; when using, add 50μl vitamin storage solution and 50μl trace element storage solution to 50mL culture medium respectively.

[0065] 2. Preparation of target gene:

[0066] 1. Based on the sequences described in Hollands, K., et al. Engineering two species of yeast as cell factories for 2′-fucosyllactose. Metab Eng, 2019., Beijing Qingke Xinye Biotechnology Co., Ltd. was commissioned to synthesize the optimized lactose transporter (LAC12) encoding gene klLac12, GDP-mannose dehydratase (GMD) gene maGMD and GDP-fucose synthase (GMER) gene hpGMER.

[0067] 2. According to the nucleotide sequence of α-1,2-fucosyltransferase (FutC) from Helicobacter pylori described in Hollands, K., et al. Engineering two species of yeast as cell factories for 2′-fucosyllactose. Metab Eng, 2019., after codon optimization, Beijing Qingke Xinye Biotechnology Co., Ltd. was commissioned to synthesize the optimized fucosyltransferase gene futC, and then the optimized synthesized Ub tag was 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 amplified by PCR using the genome of strain PO1f as a template.

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

[0070] >SEQ ID NO.1

[0071]

[0072] Table 1 Primer sequences and knockout site sequences

[0073]

[0074]

[0075] 3. Construction of plasmid:

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

[0077] Plasmid pLEP-futC-GGL was mainly constructed by fusion PCR, and the klLAC12, maGMD and hpGMER genes and the promoters and terminators used for the regulation of the above genes were amplified respectively, and the expression cassettes of the above genes were amplified by fusion PCR respectively; using the pLEP-leu plasmid as the backbone, the futC gene was cloned into the UT8 promoter of the pLEP-leu plasmid by conventional Gibson assembly, and then the klLAC12, maGMD and hpGMER gene expression cassettes were cloned into the pLEP-futC plasmid by Gibson assembly.

[0078] The construction method of plasmid pLEP-Ub-futC-GGL is to firstly obtain the optimized Ub tag and futC gene by PCR amplification, and then realize the fusion of the two genes by fusion PCR. Then, the obtained fusion gene is cloned into pLEP-leu plasmid by Gibson assembly to obtain pLEP-Ub-futC plasmid, and then the klLAC12, maGMD and hpGMER gene expression frames are cloned into pLEP-Ub-futC plasmid.

[0079] 2. Construction of plasmid pULUF-GG

[0080] Plasmid pULUF-GG was mainly constructed by fusion PCR. First, futC and Ub genes were amplified to construct the fusion protein encoding gene. Then, klLAC12, maGMD and hpGMER genes and the promoters and terminators used for the regulation of the above genes were amplified respectively. The expression frames of the above genes were amplified by fusion PCR respectively, and the vector skeleton containing the screening marker Ura3 gene expression frame was amplified, wherein the Ura3 gene expression frame had LoxP sites upstream and downstream respectively. Subsequently, the above gene expression frames were connected together by Gibson assembly to obtain the pULUF-GG plasmid.

[0081] 3. Construction of plasmid pHLUF-GG

[0082] Plasmid pHLUF-GG was obtained by replacing the Ura3 selection marker gene of the pULUF-GG plasmid with the hyg gene. Primers were designed using the pULUF-GG plasmid as a template to amplify a fragment that did not contain the Ura3 selection marker gene expression frame, and at the same time amplify the expression frame containing the hyg gene. The pHLUF-GG plasmid was constructed by Gibson assembly.

[0083] 4. Construction of plasmid pMAN

[0084] Using the genome of PO1f strain as a template, the PMI40, SEC53 and PSA1 genes were amplified respectively, 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 skeleton containing the leu gene expression cassette was amplified simultaneously. 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 through PCR and Gibson assembly. The genome of PO1f strain was used as a template to amplify the YHT1 gene and construct it at the Bsp119I restriction site of the plasmid to construct the expression plasmid pTrp-YHT1.

[0087] 6. Construction of plasmid pCas9-YHT3

[0088] The 20 bp sequence near the knockout site of the selected YHT3 gene was constructed onto a plasmid containing the Cas9 gene by gene synthesis to obtain the pCas9-YHT3 plasmid.

[0089] Example 2 Construction of an engineered strain of Yarrowia lipolytica that produces high 2′-fucosyllactose

[0090] 1. Construct a Yarrowia lipolytica strain capable of producing 2′-fucosyllactose, transform the plasmids pLEP-futC-GGL and pLEP-Ub-futC-GGL into the Yarrowia lipolytica strain PO1f, respectively, so that the Yarrowia lipolytica can synthesize GDP-L-fucose in the cell and transport lactose into the cell, so that it can synthesize 2′-FL. The specific method is as follows: (1) After Yarrowia lipolytica PO1f is cultured overnight in YPD liquid medium (containing 2% tryptone, 1% yeast extract and 2% glucose), the competent cells are prepared by the conventional yeast lithium acetate competent preparation method. (2) Add 1 μl (free plasmid) / 10 μl (linearized plasmid) plasmid to 40 μl of competent cells, and then add 2 μl salmon sperm DNA, and culture in a 30°C 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 (40 mM) to the above system, and culture in a 30°C water bath for 1 hour. (4) Add 40 μl of DMSO (final concentration of about 40%) to the above system, and heat shock at 39°C for 10 minutes. (5) Add 600 μl of Lithium Acetate (0.1M pH 6.0) and leave at room temperature for 15 minutes. (6) Take 200 μl of the above mixture and spread it on SD-ura+leu screening plates, and culture at 30°C for 2-3 days. (7) Randomly pick transformants and detect 2′-fucosyllactose in the transformants. The transformant and the original strain were fermented in YPD liquid (80g / L glucose) medium supplemented with 0.5% lactose, and the fermentation broth was taken for detection after 144 hours. The detection was mainly carried out by high performance liquid chromatography using Rezex ROA Organic Acid H+(8%) Column. The HPLC instrument used was purchased from Shimadzu Corporation. The specific conditions were: 5mM sulfuric acid was used as the mobile phase, the column temperature was set to 65°C, the flow rate was 0.6mL / min, and the injection volume was 10μL. The method for detecting the intracellular 2′-FL production is: centrifugation at 12000rpm to collect the bacterial cells, washing them twice with distilled water, and then grinding and crushing them at high speed. The resulting cell lysate was tested to measure the intracellular 2′-FL production. The results showed that the production of 2′-FL in the pLEP-futC-GGL transformed strain was 0.10 g / L, while the production 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. Construct a Yarrowia lipolytica strain capable of stably producing 2′-fucosyllactose. Transform the linearized pULUF-GG plasmid into the Yarrowia lipolytica strain PO1f and integrate it into the genome of PO1f. The specific transformation method is the same as described above. The results showed that 2'-FL could be detected in the fermentation broth of the transformant strain, and the yield of the recombinant strain F1-4 reached 0.71 g / L. Figure 1 However, the original strain PO1f did not produce 2′-fucosyllactose.

[0092] 3. Use the recombinant Yarrowia lipolytica F1-4 strain as the starting strain, remove the Ura3 gene expression frame by Cre recombinase, and then transform the linearized pULUF-GG plasmid into the F1-4 strain from which the Ura3 gene expression frame has been removed. The specific transformation method is the same as described above. Pick the transformants and ferment them in YPD liquid medium, and detect 2′-FL after 144 hours. The 2′-fucosyllactose detection method is the same as described above. The results showed that the 2′-FL production in the transformant strains was higher than that in the F1-4 strain, among which the 2′-FL production in the F2-4 strain was the highest, reaching 0.85 g / L. Figure 2 shown.

[0093] 4. Using the recombinant Yarrowia lipolytica F2-4 strain 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. The transformants were picked up and fermented in YPD liquid medium, and 2′-FL was detected after 144 hours. The 2′-fucosyllactose detection method is the same as described above. The results showed that the 2′-FL production in the F3 batch transformant strains was higher than that in the F2-4 strain, among which the 2′-FL production in the F3-5 strain was the highest, reaching 1.13 g / L. Figure 3 shown.

[0094] 5. The synthesis of 2′-FL requires GDP-L-fucose as a direct precursor, and GDP-L-fucose is catalyzed by two enzymes using GDP-D-mannose as a precursor. The recombinant Yarrowia lipolytica F3-5 strain was used as the starting strain, and the linearized pMAN plasmid was transformed into the F3-5 strain to enhance the supply of the precursor substance GDP-D-mannose, thereby promoting the accumulation of GDP-L-fucose and further promoting the synthesis of 2′-FL. The specific transformation method is the same as described above. The transformants were picked and fermented in YPD liquid medium, and 2′-FL was detected after 144 hours. The 2′-FL detection method is the same as described above. The results showed that the 2′-FL yield in the F4-2 strain was the highest, reaching 1.84 g / L. Figure 4 shown.

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

[0096] 7. Using the recombinant Yarrowia lipolytica F4-2 strain as the starting strain, the Trp gene on the genome was first knocked out by the CRISPR / Cas9 method, and then the linearized plasmid pTrp-YHT1 was transformed into the obtained Trp gene knockout strain. The transformants were selected for fermentation verification in YPM liquid medium. The results showed that the OD of the transformant MF1 600 and 2′-FL production was significantly higher than that of the F4-2 strain. Figure 6 As shown, it was shown that overexpression of YHT1 could effectively promote the absorption and utilization of mannitol by Yarrowia lipolytica strains, and further promote the accumulation of 2′-FL.

[0097] 8. In order to further promote the production level of GDP-D-mannose in cells, the linearized pTrp-SP plasmid was transformed into the MF1 strain to further enhance the synthesis pathway of GDP-D-mannose. The specific transformation method is the same as described above. The transformants were selected and fermented in YPM liquid medium, and 2′-FL was detected after 144 hours. The detection method of 2′-fucosyllactose was the same as described above. The results showed that the 2′-FL production in the MF2 strain was the highest, reaching 7.68 g / L. Figure 6 shown.

[0098] 9. In Yarrowia lipolytica cells, there are some transporters that may inhibit the absorption of mannitol. Therefore, the gene YHT3 encoding the hexose transporter yht3 that may inhibit the absorption of mannitol was knocked out. The recombinant Yarrowia lipolytica MF2 strain was used as the starting strain, and the YHT3 gene on the genome was knocked out by the CRISPR / Cas9 method. The transformants were selected for fermentation verification in YPM liquid medium. The results showed that the 2′-fucosyllactose production of the transformant MF3 was significantly increased to 9.03g / L. Figure 6 shown.

[0099] Example 3 Continuous fed-batch fermentation of MF3 strain in YPM medium

[0100] The recombinant strain MF3 capable of high-yield 2′-FL constructed in Example 2 was subjected to a batch fed fermentation experiment in a 5L fermentor. The culture medium selected in this example was YPM culture medium (2% tryptone, 1% yeast extract, 6% mannitol, 0.7% lactose), with an initial volume of 3.6L. The bacterial solution was taken out from the glycerol seed tube and streaked on a YPD solid plate and cultured for 24h. The grown monoclonal was inoculated into a shake flask containing 50mL YPD culture medium, cultured at 30°C and 220rpm for 24h, and then the seed solution was transferred to a shake flask containing 200mL YPD culture medium and continued to be cultured for 24h. After that, the seed was inoculated into a 5L fermentor. The set temperature of the fermentor was 30°C, the ventilation was 1.5v / v / m, the stirring speed was set to 750rpm, and as the dissolved oxygen concentration continued to increase, the rotation speed dropped to 500rpm, the ventilation dropped to 1v / v / m, and the pH was controlled at 5.5. The mannitol concentration in the fermenter was tested by sampling every 6 hours, and mannitol was added in batches after the initial mannitol in the culture medium was exhausted. The biomass, lactose consumption and 2′-FL production in the fermenter were tested by sampling every 6 hours. Figure 6 The fermentation results show that after 144 h of fermentation, the 2′-FL production reached the highest level, which was 30.09 g / L. Figure 7 shown.

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

[0102] Considering the high cost of YPM culture medium, the recombinant strain MF3 capable of high production of 2′-FL constructed in Example 2 was subjected to batch feeding fermentation experiment in a 5L fermenter using cheap inorganic salt culture medium. The culture medium selected in this example is CM1 culture medium (specific components as shown above, 7% mannitol, 0.7% lactose), with an initial volume of 3.6L. Take out the bacterial solution from the glycerol seed tube and streak it on the YPD solid plate and culture it for 24h. Inoculate the grown monoclonal clone into a shake flask containing 50mL YPD culture medium, culture it at 30°C and 220rpm for 24h, then transfer the seed solution to a shake flask containing 200mL YPD culture medium and continue to culture it for 24h. Then inoculate the seed into a 5L fermenter. The set temperature of the fermenter was 30°C, the ventilation was 1.5 v / v / m, and the stirring speed was set to 750 rpm. As the dissolved oxygen concentration continued to increase, the speed was reduced to 500 rpm, the ventilation was reduced to 1 v / v / m, and the pH was controlled at 5.5. Samples were taken every 6 hours to detect the mannitol concentration in the fermenter, and mannitol was added in batches after the initial mannitol in the culture medium was exhausted. Samples were taken every 6 hours to detect the biomass, lactose consumption, and 2′-FL production in the fermenter. Figure 6 The fermentation results show that after 158 h of fermentation, the 2′-FL production reached the highest level, which was 27.55 g / L. Figure 8 shown.

[0103] Matters not covered by the present invention are known technologies.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A genetically engineered bacterium for producing 2'-fucosyllactose based on mannitol, characterized in that: The genetically engineered bacteria is obtained by using Yarrowia lipolytica having the ability to produce 2'-fucosyllactose as a starting strain, overexpressing the coding genes of hexose transporter Yht1, phosphomannose mutase and 1-phosphomannose guanosyltransferase, and knocking out the coding gene of hexose transporter Yht3; Among them, the Yarrowia lipolytica with the ability to produce 2'-fucosyllactose is a process strain in the engineered strain for constructing a high-yield 2'-fucosyllactose in CN116622534A, and the process strain can express lactose transporter, GDP-mannose dehydratase, GDP-fucose synthase and α-1,2-fucosyltransferase fused with a Ub tag, and can enhance the GDP-D-mannose synthesis pathway by overexpressing 6-phosphomannose isomerase, phosphomannose mutase and 1-phosphomannose guanyl transferase using a strong promoter.

2. The genetically engineered bacterium according to claim 1, characterized in that The coding gene of the hexose transporter Yht1 is the YHT1 gene, and the gene number is YALI1_C08523g.

3. The genetically engineered bacterium according to claim 1, characterized in that The coding gene of the phosphomannose mutase is the SEC53 gene, and the gene number is YALI0_D13112g.

4. The genetically engineered bacterium according to claim 1, characterized in that The coding gene of the 1-phosphomannose guanosyltransferase is the PSA1 gene, and the gene number is YALI0_C06490g.

5. The genetically engineered bacterium according to claim 1, characterized in that The coding gene of the hexose transporter Yht3 is the YHT3 gene, and the gene number is YALI1_F25587g.

6. The method for constructing the genetically engineered bacteria according to any one of claims 1 to 5, characterized in that: The construction method comprises: constructing an integrative plasmid containing a hexose transporter Yht1 gene YHT1 expression frame and linearizing it, integrating the linearized plasmid into the genome of Yarrowia lipolytica having 2'-fucosyllactose production ability through an iterative integration method, and obtaining a first Yarrowia lipolytica strain engineered bacterium; An integrative plasmid containing expression frames of the phosphomannose mutase encoding gene SEC53 and the 1-phosphomannose guanosyltransferase encoding gene PSA1 is constructed and linearized, and transformed into the first Yarrowia lipolytica engineered bacteria to obtain a second Yarrowia lipolytica engineered bacteria; An episomal plasmid for knocking out the hexose transporter Yht3 gene YHT3 was constructed and transformed into the second Yarrowia lipolytica engineered bacteria to obtain the result.

7. The construction method according to claim 6, characterized in that: The construction method comprises: The linearized pTrp-YHT1 plasmid was transformed into Yarrowia lipolytica with Trp gene knocked out and capable of producing 2'-fucosyllactose to obtain the first Yarrowia lipolytica strain engineered bacteria; wherein the pTrp-YHT1 plasmid is an integrative plasmid containing the expression frame of the gene YHT1 encoding the hexose transporter Yht1. The linearized pTrp-SP plasmid is transformed into the first Yarrowia lipolytica strain engineered bacteria in which the Trp gene is knocked out to obtain the second Yarrowia lipolytica strain engineered bacteria; wherein the pTrp-SP plasmid is an integrative plasmid containing the expression frames of the phosphomannose mutase encoding gene SEC53 and the 1-phosphomannose guanosyltransferase encoding gene PSA1; pCas9-YHT3 is transformed into a second Yarrowia lipolytica strain engineered bacterium to obtain the genetically engineered bacterium; wherein the pCas9-YHT3 plasmid is an episomal knockout plasmid with a YHT3 gene knockout site sequence.

8. Use of the genetically engineered bacteria according to any one of claims 1 to 5 in producing 2'-fucosyllactose; further, the use is specifically: producing 2'-fucosyllactose using mannitol as a carbon source.

9. A method for producing 2'-fucosyllactose based on mannitol, characterized in that: The method comprises: adding the genetically engineered bacteria into a culture medium containing mannitol for fermentation and culturing, and separating and purifying to obtain 2'-fucosyllactose.

10. The method according to claim 9, characterized in that The culture medium components containing mannitol include: 6-8% mannitol, 2-4% tryptone, 1-2% yeast extract and 0.4-1% lactose.

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