A genetically engineered yeast strain for synthesizing disaccharides

Abstract

The present invention belongs to the field of bioengineering, genetic engineering technology and oligosaccharide (rare sugar) synthesis technology, and specifically relates to a method for producing lactose-N-disaccharide using genetically modified recombinant yeast. The technical scheme of the present invention is: a recombinant yeast cell is introduced with (1) a heterologous lactose-N-disaccharide phosphorylase ( LNBP ) gene, and (2) fructose-1-phosphate phosphorylase ( YQAB ) gene, and further preferably, also includes (3) destruction or modification of a marker gene. The recombinant yeast cell has the ability to synthesize lactose-N-biose (Lacto-N-biose, LNB) from lactose, and the yield of LNB is greatly improved by a molecular switch.

Description

Technical Field

[0001] The present invention belongs to the fields of bioengineering, genetic engineering technology and oligosaccharide (rare sugar) synthesis technology, and specifically relates to a method for producing lactose-N-disaccharide by using genetically modified yeast genetic engineering bacteria. Background Art

[0002] Breast milk is the most ideal natural "functional food" for infants and the "gold standard" for infant nutrition. Compared with breastfed infants, bottle-fed infants have a lower survival rate and a higher chance of infection, which has aroused scientists' interest in studying the composition of breast milk. Since the 21st century, researchers have found that the concentration of total HMOs (acidic and neutral oligosaccharides) in human colostrum is as high as 24g / L, but it decreases with the extension of lactation period; it is also found that HMOs are very important for the growth and development of infants and their health in the later period. It can promote the balance of infant intestinal microecology, promote the proliferation of beneficial bacteria in the intestine, inhibit the growth of harmful bacteria, resist pathogen infection, regulate the immune system and promote the cognitive development of infants. Many beneficial effects of breast milk are attributed to human milk oligosaccharides (HMOs).

[0003] With the continuous advancement of analytical tools and technologies for structural characterization, scientists are working to identify the components of breast milk, especially the rich and diverse HMOs, of which more than 200 have been identified. Among them, 2-fucosyllactose (2'-FL), lactose-N-triose (Lacto-N-tetriaose II, LNTII), lactose-N-tetraose (Lacto-N-tetraose, LNT) and lactose-N-neotetraose (LNnT) were found to be the main components of HMOs, playing the role of bifidus factors. Studies have shown that adding HMOs to infant formula milk powder with cow's milk or goat's milk as the main raw material and feeding infants can approach the effect of breastfeeding in terms of nutrition and immunity of infants. Therefore, adding HMOs with physiological functions to infant formula, some artificial milk, dairy products, baby food, functional health products or special population foods will greatly improve the physical fitness of the corresponding population and improve their quality of life, which has obvious social and economic value. Infant formula and dietary supplements added with HMOs have been sold in the markets of the United States, the European Union, Australia, New Zealand, etc. Currently, the European Union EFSA and the US FDA have approved the marketing of lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT).

[0004] Lactose-N-biose (LNB, Galβ1-3GlcNAc, chemical structure see formula 1) is a type of HMOs and the core structure of neutral HMOs. It is an important precursor for the synthesis of other neutral HMOs. However, due to its limited sources, it is urgent to establish a simple, efficient and economical synthesis pathway for obtaining the core structure lactose-N-biose (LNB) in large quantities. LNB is a product of galactose-1-phosphate and acetylglucosamine (GlcNAc) through 1,3- β In recent years, researchers have been optimizing the production and preparation methods of LNB, which is a disaccharide connected by a bond. Among the existing preparation methods, extraction from human breast milk has high production costs, low yields, and high prices. Although chemical synthesis can obtain oligosaccharides with a certain structure, it requires the participation of heavy metals, is environmentally unfriendly, and has food safety issues, which limits the commercial progress of chemical synthesis of LNB. Compared with chemical methods, biological methods have the advantages of simple processes, mild reaction conditions, and simple product treatment processes. Therefore, researchers have been trying to use biological methods to synthesize lactose-N-disaccharide (LNB) in order to break through bottlenecks and achieve low-cost, scalable production of LNB.

[0005] .

[0006] Nishimoto et al. (Biosci. Biotech. Bioch. 2007, 71 (8), 2101-2104.) synthesized lactose-N-disaccharide (LNB) by in vitro enzyme catalysis. They designed a one-pot multi-enzyme catalysis pathway for synthesizing LNB using sucrose and acetylglucosamine as substrates. Sucrose phosphatase was used to catalyze glucose-1-phosphate, which was then catalyzed with acetylglucosamine to generate LNB by enzyme catalysis after UDP-Gal / UDP-Glc cycle. CN201911055516.5 used a multi-enzyme catalytic system with good biosafety and wide application, and introduced three modules such as ATP regeneration cycle system into the multi-enzyme reaction system with galactose and acetylglucosamine. Compared with the previous method, the enzyme synthesis rate was increased by more than 13 times, and the synthesis of lactose-N-disaccharide and substrate utilization were improved. However, the above methods must use acetylglucosamine as a substrate, which greatly increases the raw material cost for synthesizing LNB.

[0007] The synthesis of HMOs by microbial fermentation is mostly focused on the biosynthesis technology of HMOs produced by fermentation of Escherichia coli. There are many patents (application numbers CN201611147477.8, CN201680052611.8, CN201611147478.2 and CN201510751641.5) that all use Escherichia coli as a host to produce HMOs heterologously. Compared with Escherichia coli, yeast fermentation production has food safety, high robustness, and no phage problems. It is one of the ideal hosts for large-scale industrial production of HMOs. CN202010187632.9 discloses an engineered strain of Saccharomyces cerevisiae, in which the yeast cells can produce 2'-fucosyllactose after genetic modification, which provides an important reference for the industrial production of HMOs by yeast. However, to date, there have been no reports on the synthesis of LNB using Escherichia coli or yeast. Summary of the invention

[0008] Purpose of the invention: To provide a yeast genetic engineering bacterium capable of producing lactose-N-biose, and the application of the yeast genetic engineering bacterium in synthesizing lactose-N-biose (Lacto-N-biose, LNB).

[0009] The present invention provides a new lactose-N-disaccharide synthesis pathway that does not require acetylglucosamine as a substrate, laying a foundation for large-scale industrial production of lactose-N-disaccharide, and has important economic value and social benefits. At the same time, the synthesis method of the present invention is efficient, mild, simple, easy to operate, low in cost, suitable for industrial production, and has good practical application value.

[0010] The technical solution of the present invention is:

[0011] A genetically engineered yeast having the ability to synthesize lactose-N-biose (Lacto-N-biose, LNB) using lactose as raw material.

[0012] Preferably, the lactose is used as the raw material, and a culture medium containing lactose or galactose can be selected as the raw material to synthesize LNB.

[0013] The yeast genetically engineered bacteria preferably comprises (1) a heterologous lacto-N-biose phosphorylase (Lnbp) gene and (2) a fructose-1-phosphate phosphorylase (YqaB) gene. The yeast genetically engineered bacteria has the ability to synthesize lactose-N-biose (Lacto-N-biose, LNB) from lactose without the need to introduce special transport proteins.

[0014] The lactose-N-biose phosphorylase (Lacto-N-biose phosphorylase, Lnbp), also known as 1,3-β-galactosyl-N-acetylhexosamine phosphorylase (1,3-beta-galactosyl- N -acetylhexosaminephosphorylase), EC 2.4.1.21. It comprises an enzyme having an amino acid sequence with at least 67%, at least 68%, at least 69%, 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, and has the activity of producing lactose-N-biose LNB.

[0015] The lactose-N-biose phosphorylase Lnbp is preferably derived from Bifidobacterium longum Bifidobacterium long of LNBP1 (Genbank No. BAD80751.1, PDB: 2ZUS), and its amino acid sequence is shown in SEQ ID NO: 1.

[0016] The lactose-N-biose phosphorylase Lnbp is preferably derived from Bacillus Bacillus cereus of LNBP2 (Genbank No. WP_078422288.1); its amino acid sequence is shown in SEQ ID NO:2.

[0017] As attached Figure 1 As shown, the amino acid sequence identity between SEQ ID NO: 1 and SEQ ID NO: 2 is 52.88%.

[0018] The fructose-1-phosphate phosphorylase (YqaB) comprises an enzyme having an amino acid sequence having at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:3 or SEQ ID NO:4.

[0019] The fructose-1-phosphate phosphorylase (YqaB), preferably, is derived from Escherichia coli Escherichia coli (Genbank No. APQ20426.1) EcYQAB , whose amino acid sequence is shown in SEQ ID NO:3.

[0020] The fructose-1-phosphate phosphorylase (YqaB) is preferably derived from the bacterial sweet potato stem rot fungus Dickeyadadantii (Genbank No. WP_013319095.1) DdYQAB , whose amino acid sequence is shown in SEQ ID NO:4.

[0021] As attached Figure 2 As shown, the amino acid sequence identity between SEQ ID NO:3 and SEQ ID NO:4 is 67%.

[0022] In some embodiments, yeast cells are used as hosts to introduce heterologous lactose-N-biose phosphorylase genes ( LNBP ) and fructose-1-phosphate phosphorylase gene ( YQAB ), which increased the ability of the original yeast cells to synthesize LNB.

[0023] In some embodiments, the yeast genetically engineered bacteria may be of the genus Saccharomyces ( Issatchenkia sp.), Candida spp. Candida sp.), Kluyveromyces ( Kluyveromyces sp.), Pichia Pichia sp.), Schizosaccharomyces spp. Schizosaccharomyces sp.), Hansenula Hansenula sp.), Torulella sp. Torula spore sp.), Yarrowia Yeastsp.), Zygosaccharomyces spp. Zygosaccharomyces sp.) or Saccharomyces spp. Saccharomyces sp.) yeast cells.

[0024] Further preferably, the genetically engineered yeast is Saccharomyces cerevisiae (Saccharomyces cerevisiae) 、Kluyveromyces lactis( Kluyveromyces lactis )、Kluyveromyces marxianus( Kluyveromyces marxinus) , Yarrowia lipolytica ( Yarrowia lipolytica )、Red yeast rice( Rhodotorula graminis )、Pasteurella Saccharomyces pastorianus ) and other cells; among them, Kluyveromyces lactis ( Kluyveromyces lactis ) is a yeast cell that has passed the GRAS food safety certification of the U.S. Food and Drug Administration, and is more easily accepted in food or food additive management.

[0025] The yeast strain, in a preferred embodiment, can be selected from the group consisting of: Kluyveromyces lactis ( Kluyveromyces lactis ) and Kluyveromyces marxianus ( Kluyveromyces marxianus ). The deposit number of Kluyveromyces lactis in ATCC is ATCC 8585. The deposit number of Kluyveromyces marxianus in ATCC is ATCC 46537.

[0026] Strains of the above species can also be easily obtained by the public in many domestic culture collection centers, such as the China General Microbiology Center (CGMCC), China Center for Type Culture Collection (CCTCC), Guangdong Microbiology Culture Collection Center (GDMCC), etc.

[0027] In one aspect, the genetically engineered yeast comprises (1) a lacto-N-biose phosphorylase gene ( LNBP ) introduction, (2) fructose-1-phosphate phosphorylase gene ( YQAB ) introduction of yeast marker genes, and (3) disruption or modification of yeast marker genes.

[0028] The yeast marker gene is a galactose-1-phosphate uridyl transferase gene ( WRONG Yeast marker genes ( WRONG ) is a necessary condition for achieving high-yield LNB.

[0029] The term "disruption" or "modification" means that the coding region and / or control sequence of the reference gene is partially or completely modified (e.g., by deletion, insertion and / or substitution of one or more nucleotides) so that the expression of the encoded polypeptide is absent (inactivated) or reduced, and / or the enzymatic activity of the encoded polypeptide is absent or reduced. The effect of the disruption can be measured using techniques known in the art, such as detecting the absence or reduction of galactose consumption using measurements from cell-free extracts referenced herein.

[0030] In certain embodiments, the genetically engineered yeast strain comprises disruption or modification of one or more yeast marker genes.

[0031] The yeast marker gene comprises a partial amino acid sequence of galactose-1-phosphate uridyl transferase (GalT), and has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% amino acid sequence identity to SEQ ID NO:5 or SEQ ID NO:6.

[0032] The yeast genetic engineering bacteria is preferably a lactic acid Kluyveromyces genetic engineering bacteria, and further preferably, comprises at least one yeast marker gene that is destroyed or modified, and contains the amino acid sequence of the yeast marker, and the nucleic acid sequence encoding the yeast marker. The nucleic acid sequence of the yeast marker gene has a Genebank number of X07039.1; its amino acid sequence has a Genebank number of CAA30091.1, as shown in SEQ ID NO:5.

[0033] The genetically engineered Kluyveromyces marxianus strain is further preferably one in which at least one yeast marker gene is destroyed or modified, and comprises an amino acid sequence of the yeast marker and a nucleic acid sequence encoding the yeast marker. The nucleic acid sequence of the yeast marker gene has a Genebank number of NC_036026.1; its amino acid sequence has a Genebank number of XP_022674659.1, as shown in SEQ ID NO:6.

[0034] In one aspect, a genetically engineered host cell having an active synthetic LNB pathway, wherein the cell comprises a yeast marker gene WRONG (eg, disruption or modification of SEQ ID NO: 5 and SEQ ID NO: 6). In some embodiments, when cultured under the same conditions, the production of LNB in ​​the genetically engineered host strain is significantly improved compared to the parent strain.

[0035] As attached Figure 3 As shown, the amino acid sequence identity between SEQ ID NO:5 and SEQ ID NO:6 is 79.84%.

[0036] The recombinant construction technology of the genetically engineered Kluyveromyces lactis or Kluyveromyces marxianus comprises the following steps:

[0037] (1) Constructing an expression cassette and introducing the lactose-N-biose phosphorylase gene into Kluyveromyces lactis cells or Kluyveromyces marxianus cells ( LNBP ); and / or

[0038] (2) Constructing an expression cassette and introducing the fructose-1-phosphate phosphorylase gene into Kluyveromyces lactis cells or Kluyveromyces marxianus cells ( YQAB ); and / or

[0039] (3) Constructing a knockout cassette or using a point mutation kit to knock out or modify yeast marker genes in Kluyveromyces lactis cells or Kluyveromyces marxianus cells ( WRONG ).

[0040] In some embodiments, the yeast genetically engineered bacteria, by introducing artificial transcription factor CamR regulation CAMO Promoter.

[0041] Preferably, the genetically engineered yeast increases the yield of LNB by regulating the pathway of GlcNAc-6-P flowing to GlcNAc-1-P.

[0042] Another embodiment of the present invention is the use of the genetically engineered yeast in synthesizing lactose-N-disaccharide (LNB).

[0043] The yeast genetic engineering bacteria is used in the synthesis of lactose-N-disaccharide (LNB), with lactose or glucose and galactose as substrates.

[0044] The application preferably comprises the step of generating an LNB.

[0045] Preferably, the fermentation product of the genetically engineered yeast may be purified by centrifugation, filtration, decolorization, nanofiltration, chromatography, crystallization, recrystallization, and the like.

[0046] In another specific embodiment of the present invention, lactose-N-disaccharide synthesized by the above technical solution is provided. In addition, based on the synthesis method of the present invention, the synthesis of all oligosaccharides or polysaccharides containing lactose-N-disaccharide as the skeleton structure is also within the protection scope of the present invention.

[0047] definition

[0048] The term "host cell" means any cell type susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector. The term "host cell" encompasses any progeny of a parent cell that is different from the parent cell due to mutations that occur during replication. The term "genetically engineered bacteria" is defined herein as a non-naturally occurring host cell that includes one or more (e.g., two, several) heterologous polynucleotides. The host cell can be a mutant strain described herein, or can be further disrupted to provide a mutant strain described herein.

[0049] Heterologous polynucleotide: The term "heterologous polynucleotide" is defined herein as a polynucleotide that is not native to the host cell; a native polynucleotide to which structural modifications have been made to the coding region; a native polynucleotide whose expression is quantitatively altered due to manipulation of DNA by recombinant DNA techniques, e.g., a different (foreign) promoter; or a native polynucleotide in a host cell that has one or more additional copies of the polynucleotide to quantitatively alter expression. A "heterologous gene" is a gene that includes a heterologous polynucleotide.

[0050] Sequence identity: The degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". For the purposes described herein, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443-453), as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., Trends Genet., 2000, 16:276-277), preferably version 3.0.0 or higher.

[0051] Beneficial Effects

[0052] The present invention uses Kluyveromyces genetically engineered bacteria for the first time, without the addition of coenzyme factors such as ATP and acetylglucosamine (GlcNAc), and only uses cheap carbon sources such as lactose to achieve efficient synthesis of LNB. It is superior to in vitro enzyme catalysis in terms of food safety, energy utilization and production cost. This is also the first time that LNB has been synthesized by microbial fermentation. The product has high catalytic efficiency and conversion rate, while low waste emissions, which is beneficial to the downstream purification effect of the product and makes it easier to obtain high-purity LNB products.

[0053] The technology of the present invention has positive significance for the industrial production of human milk oligosaccharide LNB. The LNB obtained by this method is green and safe, and can be produced efficiently and sustainably on a large scale. It has important practical value and broad development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 . Comparison result of amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2.

[0055] Figure 2 . Comparison results of the amino acid sequences shown in SEQ ID NO:3 and SEQ ID NO:4.

[0056] Figure 3 .Comparison results of the amino acid sequences shown in SEQ ID NO:5 and SEQ ID NO:6.

[0057] Figure 4 . is the HPLC spectrum of the fermentation product of StrainKL-LNB-4.

[0058] Figure 5 .LC-MS image of LNB standard.

[0059] Figure 6 . LC-MS chart of the product at rt=10.5 min in the HPLC chart. DETAILED DESCRIPTION

[0060] The present invention is further described in detail below through examples. It should be understood that the specific implementation methods described herein are only used to explain the present invention and are not used to limit the present invention. Modifications or replacements to the details and forms of the technical solution without departing from the structural concept and scope of use of the present invention all fall within the protection scope of the present invention.

[0061] It should be noted that the terms used are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. In addition, it should also 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. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are to describe specific embodiments, rather than to limit the scope of protection of the present invention. If the experimental methods of specific conditions are not indicated in the following specific embodiments, the conventional methods and conditions of molecular biology within the art are generally followed, and such techniques and conditions are fully explained in the literature. See, for example, Sambrook et al., the techniques and conditions described in "Molecular Cloning: A Laboratory Manual", or according to the conditions recommended by the manufacturer.

[0062] In the following specific embodiments, if the experimental methods for specific conditions are not specified, they are usually based on conventional methods and conditions of molecular biology within the technology of the field, and such techniques and conditions are fully explained in the literature; all materials, reagents, etc., unless otherwise specified, can be obtained from commercial channels.

[0063] In the following examples, the Kluyveromyces lactis has an accession number of ATCC 8585; the Kluyveromyces marxianus has an accession number of ATCC 46537.

[0064] In the following examples, all high-fidelity enzymes used in the PCR amplification process were purchased from Novozyme Biotech Co., Ltd. 2* MultiF Seamless Assembly Mix was purchased from ABclonal Technology Biotech Co., Ltd.

[0065] In the following examples, the PCR amplification system is Phanta Super-Fidelity DNA Polymerase 1 μL, 2×Phanta MaxBuffer 25 μL, dNTPs (10 mM) 1 μL, primers F and R 2 μL each, template DNA 1 μL, and ultrapure water is added to a total volume of 50 μL. The PCR amplification program is 95°C pre-denaturation for 3 min, 95°C denaturation for 15 s, 54°C annealing for 30 s, 72°C extension for 2-3 min, 2-4 step cycles for 30 times, 72°C final extension for 5 min, and finally stored at 4°C.

[0066] In the following examples, the target gene bands recovered by agarose gel electrophoresis were ligated using 2*MultiFSeamless Assembly Mix, and the ligation system was X μL of DNA fragment (0.5-1 pmol), 5 μL of 2*MultiFSeamless Assembly Mix, and ultrapure water was added to a total volume of 10 μL.

[0067] Preferably, the genetically engineered Kluyveromyces yeast can transfer lactose into cells, and degrade it into galactose and glucose in the cells. In addition to entering glycolysis and being used for yeast growth and energy metabolism, glucose is also catalyzed by multi-step reactions to generate acetylglucosamine-6-phosphate, which is catalyzed by fructose-1-phosphate phosphorylase (YqaB) to acetylglucosamine; while galactose is catalyzed to galactose-1-phosphate, and finally galactose-1-phosphate and acetylglucosamine are synthesized into LNB by lactose-N-disaccharide phosphorylase (Lnbp).

[0068] In the following examples, the yeast transformation method is as follows:

[0069] (1) Prepare yeast competent cells: Take a small amount of frozen yeast strains and streak them on a plate of solid culture medium, invert and culture at 30°C for 2 days. Pick a single yeast colony in 50 mL of liquid culture medium and culture at 30°C, 220 rpm until OD 600 Between 0.8-1.5. Collect the cells and wash with 25 mL sterile water, centrifuge at 1500 × g for 10 min at room temperature, and discard the supernatant. Add 1 mL 100mM lithium chloride buffer, resuspend the precipitate, centrifuge at 12000 rpm for 30s, and discard the supernatant. Add 400 μL 100 mM lithium chloride buffer again, resuspend the precipitate, obtain the competent yeast cells, and divide them into 50 μL / tube for transformation. At the same time, boil 1 mL salmon sperm DNA for 5 min and quickly ice bath to prepare single-stranded DNA.

[0070] (2) Transformation: Centrifuge the competent yeast prepared above and remove the residual lithium chloride solution with Tips. For each transformation, add the following in the following order: 50% PEG3350 (240 μL); 1M LiCl (36 μL); 2 mg / mL single-stranded Salmon sperm DNA (25 μL); 5-10 μg / 50 μL H2O plasmid DNA (50 μL), vortex vigorously until the precipitated bacteria are completely distributed; incubate in a 30 ℃ water bath for 30 min; heat shock in a 42 ℃ water bath for 20-25 min; collect the yeast after centrifugation at 8000 rpm for 10 min; then, resuspend the yeast in 500 μL liquid culture medium and incubate in a shaking incubator at 30 ℃; after 1-4 h, take 25-100 μL of bacterial solution and spread it on a selective culture medium plate and incubate it upside down at 30 ℃.

[0071] In the following examples, the yeast genome extraction method is as follows: a single colony of Kluyveromyces was picked into 1 mL YPD (10 mL centrifuge tube) medium, and cultured overnight at 30°C and 200 rpm; 600 μL of bacterial solution was taken into a 1.5 mL EP tube, centrifuged at 10,000 rpm for 1 min, the supernatant was removed, and then 600-800 μL of genome extraction buffer and 100 μL of quartz sand were added, and the mixture was fully shaken for 5 min. In a 65°C water bath for 30 min, the mixture was inverted every 10 min. The supernatant was taken into a new 1.5 mL EP tube, an equal volume of DNA extraction solution was added, the mixture was mixed by pipetting, and the mixture was centrifuged at 13,000 rpm for 10 min. 400 μL of supernatant was taken into a new 1.5 mL EP tube, 0.6 times the volume of isopropanol and 40 μL of 3M sodium acetate were added, the mixture was mixed, and the mixture was allowed to stand at -20°C for 30 min. Centrifuged at 13,000 rpm for 10 min, the supernatant was discarded, and the yeast genome was obtained. Wash twice with 70% ethanol, dissolve in ddH2O after the ethanol evaporates, and store at -20℃.

[0072] In the following example, high performance liquid chromatography (HPLC) was used to analyze the fermentation reaction products of genetically engineered yeast under the following conditions: chromatographic column model: Aminex HPX-87H (300×7.8 mm), mobile phase was 5 mM H2SO4 aqueous solution, column temperature was 45°C, injection volume was 10 μL, UV detector (Hitachi Chromaster), wavelength was 210 nm, flow rate was 0.5 mL / min.

[0073] The fermentation product of the yeast genetically engineered bacteria of the present invention can be purified by centrifugation, filtration, decolorization, nanofiltration, chromatography purification and crystallization to obtain a reaction solution or solid with high purity; it can also be made into powder by spray drying or freeze drying technology.

[0074] Example 1. Preparation of genetically engineered Kluyveromyces lactis

[0075] 1. Yeast marker genes KL-ΔGALT::loxp-Kan-loxp Construction of knockout cassette

[0076] according to WRONG Upstream and downstream sequences, PUG6 plasmid and resistance screening CAN The sequence information of the amplification primers was designed as shown in Table 1. The primers were designed using the Kluyveromyces lactis genome as a template.

[0077] galt up-s: 5'-cactatagaacgcaaataccattgacatgacagat-3';

[0078] galt up-a: 5'-gttgtcgacctgccttgatgaaataaattgggcac-3';

[0079] galt down-s: 5'-tccactagtgTGAgcaggcaaagcagaaaata-3';

[0080] galt down-a: 5'-cactataggggagagaatttgaattccatttgagaactg-3',

[0081] After PCR amplification with the above primers, WRONG Upstream and downstream sequences.

[0082] The resistance sequence Kan was obtained by PCR amplification using the pUG6 plasmid containing Kan resistance as a template and primers KanMX4-galt-s: 5'-atttcatcaaggcaggtcgacaacccttaat-3' and KanMX4-galt-a: 5'-tgctttgcctgcTCAcactagtggatctgatatcacc-3'. The PUG6 plasmid backbone was obtained by PCR amplification using the PUG6 plasmid as a template and primers PUG6-galt-s: 5'-tggaattcaaattctctccctatagtgagtcgtatt-3' and PUG6-galt-a: 5'-caatggtatttgcgttctatagtgtcacctaaatcg-3'.

[0083] After amplification, perform agarose gel electrophoresis, recover the target bands that meet the expected size, and connect them using 2*MultiF Seamless Assembly Mix. Add each fragment in equal moles. Connect at 50℃ for 30 minutes and then transform E. coli. Pick the positive E. coli transformants for plasmid extraction, and name the plasmid PUG6-GALT. Use the plasmid as a template, galt up-s and galt down-a as primers, and perform PCR amplification with the same sequence as above to obtain KL-ΔGALT:: loxp-Can-loxp Knockout box.

[0084] 2. KL-ΔGALT::lnbp1-loxp-Kan-loxp Expression cassette construction

[0085] according to LNBP1 sequence, LNBP1 The promoter and terminator sequence information and PUG6-GALT plasmid information were used to design amplification primers as shown below:

[0086] Lnbp1-s(Lac4Pro):5'-actgaaagatATGACTAGCACCGGCCGCT-3';

[0087] Lnbp1-a: 5'-tggttacaacaTCAGGCTTCACGCCAGGCGA-3';

[0088] Lac4Pro-s(lnbp1 / 2):5'-catcaagcatatgtatcacgttgacttgg-3';

[0089] Lac4Pro-a(lnbp1):5'-CGGTGCTAGTCATctttcagttctcgatgag-3';

[0090] Ter-lnbp1-s: 5'-CGTGAAGCCTGAtgttgtaaccagtgtgcgc-3';

[0091] Ter-lnbp / 2-a: 5'-cgacctggaaaattattggaatattacgaggat-3';

[0092] PUG6-galt-lnbp1 / 2-s: 5'-attccaataattttccaggtcgacaacccttaata-3';

[0093] PUG6-galt-lnbp1 / 2-a:5'-gtcaacgtgatacatatgcttgatgaaataaattgggcac-3'.

[0094] Synthesized and codon-optimized LNBP1 The coding sequence was used as a template, Lnbp1-s (Lac4Pro) and Lnbp1-a were used as primers, and PCR amplification was performed to obtain LNBP1 The sequence was obtained by PCR amplification using the Kluyveromyces lactis genome as template and Lac4Pro-s (lnbp1 / 2) and Lac4Pro-a (lnbp1) as primers. LNBP1 The promoter of Kluyveromyces lactis genome was used as template and Ter-lnbp1-s and Ter-lnbp / 2-a were used as primers to obtain the LNBP1 The PUG6-GALT plasmid was used as a template and PUG6-galt-lnbp1 / 2-s and PUG6-galt-lnbp1 / 2-a were used as primers to obtain the PUG6-GALT plasmid backbone by PCR amplification. Agarose gel electrophoresis was performed as above to recover LNBP1After the promoter, terminator sequence and PUG6-GALT plasmid backbone band were identified, the fragments with equal molar numbers were connected using 2* MultiF Seamless Assembly Mix. After 30 minutes of connection at 50℃, E. coli transformation was performed. The positive E. coli transformants were selected for plasmid extraction and the plasmid was named PUG6-GALT-LNBP1\. PUG6-GALT-lnbp1 was used as a template and galt up-s and galt down-a were used as primers for PCR amplification to obtain KL-ΔGALT::lnbp1-loxp-Kan-loxp Expression cassette.

[0095] 3. KL-ΔGALT::lnbp2-loxp-Kan-loxp Expression cassette construction

[0096] according to LNBP2 sequence, LNBP2 The amplification primers were designed based on the promoter and terminator sequence information and PUG6-GALT plasmid information:

[0097] Lnbp2-s(Lac4Pro):5'-ctgaaagatATGAAGAAGAAGAAGGGTAGAGT-3';

[0098] Lnbp2-a: 5'- ActggttacaacaTTAACCTTCATTAGAAACATCTTCCCA-3';

[0099] Lac4Pro-a(lnbp2):5'-ACCCTTCTTCTTCTTCATctttcagttctcgatgagt-3';

[0100] Ter-lnbp2-s: 5'-CTAATGAAGGTTAAtgttgtaaccagtgtgcgc-3'.

[0101] Synthesized and codon-optimized LNBP2 The coding sequence was used as a template, Lnbp2-s (Lac4Pro) and Lnbp2-a were used as primers, and PCR amplification was performed to obtain LNBP2 The sequence was obtained by PCR amplification using the Kluyveromyces lactis genome as a template and Lac4Pro-s (lnbp1 / 2) and Lac4Pro-a (lnbp2) as primers. LNBP2The promoter of . Using the Kluyveromyces lactis genome as a template, Ter-lnbp / 2-a and Ter-lnbp2-s as primers, PCR amplification was performed to obtain the terminator sequence of lnbp2. Using the PUG6-GALT plasmid as a template, PUG6-galt-lnbp1 / 2-s and PUG6-galt-lnbp1 / 2-a as primers, PCR amplification was performed to obtain the PUG6-GALT plasmid backbone. Agarose gel electrophoresis was performed in the same manner as above. LNBP2 The promoter, terminator sequence and PUG6-GALT plasmid backbone band were recovered and connected. Transformed into E. coli. Positive E. coli transformants were selected for plasmid extraction, and the plasmid was named PUG6-GALT-LNBP2. PUG6-GALT-LNBP2 was used as a template and galt up-s and galt down-a were used as primers for PCR amplification to obtain KL-ΔGALT:LNBP2-loxp-Kan-loxp Expression cassette.

[0102] 4. KL-ΔXYL1::LNBP1-loxp-Kan-loxp Expression cassette construction

[0103] according to XYL1 Upstream and downstream sequences, PUG6 plasmid, Kan for screening resistance and LNBP1 The sequence information of the expression cassette and the amplification primers were designed as follows:

[0104] xyl1 up-s:5'-gtgacactatagactcgagcttgaaacttcga-3';

[0105] xyl1 up-a:5'-gttgtcgacctgctcaccattattgcggaagag-3';

[0106] xyl1 down-s: 5'-gatccactagtgAACTTTTCAGGTGCGTTGAT-3';

[0107] xyl1 down-a: 5'-ctatagggagaaggtgagtggcatcaattatg-3';

[0108] KanMX4-xyl1-s: 5'-caataatggtgagcaggtcgacaacccttaat-3';

[0109] KanMX4-xyl1-a: 5'-acCTGAAAAGTTcactagtggatctgatatcacc-3';

[0110] PUG6-xyl1-s: 5'-tgccactcaccttctccctatagtgagtcgta-3';

[0111] PUG6-xyl1-a:5'-caagctcgagtctatagtgtcacctaaatcgtat-3';

[0112] PUG6-xyl1-lnbp*2-s:5'-ttcaagcgggaacagcaggtcgacaacccttaat-3';

[0113] PUG6-xyl1-lnbp*2-a:5'-cgtgatacatatgtcaccattattgcggaagag-3';

[0114] Ter-lnbp*2-s: 5'-CGTGAAGCCTGAaaatagtttccatgtagaatttcg-3';

[0115] Ter-lnbp*2-a: 5'-acctgctgttcccgcttgaagttagc-3';

[0116] Lnbp*2-s: 5'-cgcaataatggtgacatatgtatcacgttgacttgg-3';

[0117] Lnbp*2-a: 5'-TGgaaactatttTCAGGCTTTCACGCCAGGCGATA-3'.

[0118] The Kluyveromyces lactis genome was used as template and xyl1 up-s, xyl1 up-a, xyl1 down-s, xyl1 down-a were used as primers to obtain the gene. XYL1Upstream and downstream sequences. Using the pUG6 plasmid containing Kan resistance as a template, KanMX4-xyl1-s and KanMX4-xyl1-a as primers, PCR amplification was performed to obtain the resistance sequence Kan. Using the PUG6 plasmid as a template, PUG6-xyl1-s and PUG6-xyl1-a as primers, PCR amplification was performed to obtain the PUG6 plasmid backbone. The target band obtained by agarose gel electrophoresis was connected with 2* MultiF Seamless Assembly Mix at 50°C for 30 minutes, and then Escherichia coli was transformed. The plasmid was extracted from the positive Escherichia coli transformant and named PUG6-XYL1. Using the PUG6-XYL1 plasmid as a template, PUG6-xyl1-lnbp*2-s and PUG6-xyl1-lnbp*2-a as primers, PCR amplification was performed to obtain the PUG6-XYL1 plasmid backbone. Then, the lactic acid Kluyveromyces genome was used as a template, Ter-lnbp*2-s and Ter-lnbp*2-a were used as primers, and PCR amplification was performed to obtain the terminator sequence; the PUG6-GALT-LNBP1 plasmid was used as a template, Lnbp*2-s and Lnbp*2-a were used as primers, and PCR amplification was performed to obtain lnbp1 and its promoter. LNBP1 The promoter and terminator sequence bands were recovered, connected, transformed into E. coli, and the plasmid was extracted. The plasmid was named PUG6-XYL1-LNBP1. PCR amplification was performed using the PUG6-XYL1-LNBP1 plasmid as a template and xyl1 up-s and xyl1 down-a as primers to obtain KL-ΔXYL1::LNBP1-loxp-Kan-loxp Expression cassette.

[0119] 5. KL-ΔXYL1::LNBP2-loxp-Kan-loxp Expression cassette construction

[0120] According to the relevant plasmid information, the corresponding primers are designed as follows:

[0121] lnbp2-KL-XYL1-F:5'-gagaactgaaagatATGAAGAAGAAGAAGGGTAGAG-3';

[0122] lnbp2-KL-XYL1-R:5'-atggaaactatttTTAACCTTCATTAGAAACATCTTCCCA-3';

[0123] PUG-lnbp2-XYL1-F:5'-AATGAAGGTTAAaaatagtttccatgtagaatttcg-3';

[0124] PUG6-lnbp2-XYL1-R: 5'-CTTCTCTCTCATctttcagttctcgatgagt-3'.

[0125] Using plasmid PUG6-GALT-LNBP2 as template and lnbp2-KL-XYL1-F and lnbp2-KL-XYL1-R as primers, the LNBP2 Sequence. Using plasmid PUG6-XYL1-LNBP1 as template, PUG-lnbp2-XYL1-F and PUG-lnbp2-XYL1-R as primers, synthesize the plasmid backbone part. Then perform agarose gel electrophoresis, recover the target band of the expected size, and connect each fragment using 2* MultiF Seamless Assembly Mix. After E. coli transformation, pick the positive transformant for plasmid extraction, and name the plasmid PUG6-XYL1-LNBP2. Use it as a template, xyl1 up-s and xyl1 down-a as primers for PCR amplification to obtain KL-ΔXYL1::LNBP2-loxp-Kan-loxp Expression cassette.

[0126] 6. KL-ΔXYL3::GALK2-loxp-Kan-loxp Expression cassette construction

[0127] according to XYL3 Upstream and downstream sequences, PUG6 plasmid, Kan for screening resistance and GALK2 The sequence information of the gene was used to design amplification primers as follows:

[0128] xyl3 up-s: 5'-gacactatagaacgcgtatttgttttggggctaatagt-3';

[0129] xyl3 up-a: 5'-gtcgacctgcaccaattctaatctataatttggttca-3';

[0130] xyl3 down-s: 5'-tccactagtggGTCGTATTGACAAATGTCAATATG-3';

[0131] xyl3 down-a: 5'-cgactcactataggTTAGTGTGATGACGCTTCCT-3';

[0132] KanMX4-xyl3-s:5'-gattagaattggtgcaggtcgacaacccttaata-3';

[0133] <h2 style=";text-align:left;direction:ltr">KanMX4-xyl3-a:5'-GTCAATACGACccactagtggatctgatatcac-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0134] <h2 style=";text-align:left;direction:ltr"> PUG6-xyl3-s:5'-CATCACACTAAcctatagtgagtcgtattaatttcg-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0135] <h2 style=";text-align:left;direction:ltr"> PUG6-xyl3-a:5'-acaaatacgcgttctatagtgtcacctaaat-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0136] <h2 style=";text-align:left;direction:ltr"> PUG6-xyl3-galk-s:5'-gaacgtcggtgcaggtcgacaacccttaata-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0137] <h2 style=";text-align:left;direction:ltr"> PUG6-xyl3-galk-a:5'-gtaattcgattgagccaattctaatctataatttggttca-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0138] <h2 style=";text-align:left;direction:ltr"> TefPro-s(galk):5'-agattagaattggctcaatcgaattacacagaaca-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0139] <h2 style=";text-align:left;direction:ltr"> TefPro-a(galk):5'-AGGAACGGACATttttaatgttacttctcttgcagt-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0140] <h2 style=";text-align:left;direction:ltr"> galk-s(TefPro):5'-gaagtaacattaaaaATGTCCGTTCCTATTGTGCCA-3';<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0141] <h2 style=";text-align:left;direction:ltr"> galk-a:5'-gtcgacctgcaccgacgttcattaagaatact-3'。<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0142] Using the Kluyveromyces lactis genome as a template, xyl3 up-s, xyl3 up-a; xyl3 down-s, xyl3down-a as primers, PCR amplification was performed to obtain the upstream and downstream sequences of XYL3. Using the pUG6 plasmid containing Kan resistance as a template, KanMX4-xyl3-s, KanMX4-xyl3-a as primers, PCR amplification was performed to obtain the resistance sequence Kan. Using the PUG6 plasmid as a template, PUG6-xyl3-s, PUG6-xyl3-a as primers, PCR amplification was performed to obtain the PUG6 plasmid skeleton. All target bands were connected and transformed, and the positive transformants were selected for plasmid extraction. The plasmid was named PUG6-XYL3. Using the PUG6-XYL3 plasmid as a template, PUG6-xyl3-galk-s, PUG6-xyl3-galk-a as primers, PCR amplification was performed to obtain the PUG6-Xyl3 plasmid skeleton. Subsequently, the promoter sequence was obtained by PCR amplification using the Kluyveromyces lactis genome as a template and TefPro-s (galk) and TefPro-a (galk) as primers; GALK2 and its terminator sequence were obtained by PCR amplification using the Kluyveromyces lactis genome as a template and galk-s (TefPro) and galk-a as primers. GALK The plasmid was extracted from the positive transformant and named PUG6-Xyl3-GALk. The PUG6-XYL3-GALK plasmid was used as a template and xyl3 up-s and xyl3 down-a were used as primers for PCR amplification to obtain KL-ΔXYL3:: GALK2-loxp-Kan-loxp Expression cassette.

[0143] 7. KL-ΔXYL2::YQAB-loxp-kan-loxp Expression cassette construction

[0144] according to XYL2 Upstream and downstream sequences, PUG6 plasmid, Kan for screening resistance and YQAB The sequence information of the gene was used to design the amplification primers:

[0145] xyl2 up-s: 5'-cactatagaacgcTCTTcccgtttctccatctgaat-3';

[0146] xyl2 up-a: 5'-gacctgcaTGTCTGAACCGCAGATACCG-3';

[0147] xyl2 down-s: 5'-cactagtggGATCGGTGCCAAAGAATTAC-3';

[0148] xyl2 down-a: 5'-actatagggttacctcttctcgcttcttc-3';

[0149] KanMX4-xyl2-s:5'-CGGTTCAGACAtgcaggtcgacaacccttaat-3';

[0150] KanMX4-xyl2-a: 5'-TGGCACCGATCccactagtggatctgatatcac-3';

[0151] PUG6-xyl2-s: 5'-gcgagaagaggtaaccctatagtgagtcgtattaatttcg-3';

[0152] PUG6-xyl2-a: 5'-acgggAAGAgcgttctatagtgtcaccta-3';

[0153] PUG6-xyl2-yqab-s:5'-attccaataattttctgcaggtcgacaaccct-3';

[0154] PUG6-xyl2-yqab-a:5'-cgattgagGTCTGAACCGCAGATACCG-3';

[0155] TefPro-s(yqab):5'-CTGCGGTTCAGACctcaatcgaattacacagaacac-3';

[0156] TefPro-a(yqab):5'-CGTATCTTTCGTACATttttaatgttatacttctcttgcag-3';

[0157] Ter-yqab-s: 5'-GTTGCTCGAGtgttgtaaccagtgtgcgct-3';

[0158] Ter-yqab-a:5'-gtcgacctgcagaaaattattggaatattacgaggat-3';

[0159] The Kluyveromyces lactis genome was used as template and xyl2 up-s, xyl2 up-a, xyl2 down-s, xyl2 down-a were used as primers to obtain the XYL2Upstream and downstream sequences. Using the pUG6 plasmid containing Kan resistance as a template, KanMX4-xyl2-s and KanMX4-xyl2-a as primers, PCR amplification was performed to obtain the resistance sequence Kan. Using the PUG6 plasmid as a template, PUG6-xyl2-s and PUG6-xyl2-a as primers, PCR amplification was performed to obtain the PUG6 plasmid skeleton. The target bands were connected, transformed, and the plasmids were extracted. The plasmid was named PUG6-XYL2. Using it as a template, PUG6-xyl2-yqab-s and PUG6-xyl2-yqab-a as primers, PCR amplification was performed to obtain the PUG6-XYL2 plasmid skeleton. Subsequently, the promoter sequence was obtained by PCR amplification using the Kluyveromyces lactis genome as a template and TefPro-s (yqab) and TefPro-a (yqab) as primers; the terminator sequence was obtained by PCR amplification using the Kluyveromyces lactis genome as a template and Ter-yqab-s and Ter-yqab-a as primers. YQAB The coding sequence was used as a template and PCR amplified EcYQAB or DdYQAB Sequence. The PUG6-XYL2 plasmid backbone, promoter, termination sequence and YQAB After the bands were recovered, they were connected with 2* MultiF Seamless Assembly Mix for 30 minutes and then transformed into E. coli. The positive E. coli transformants were selected for plasmid extraction and the plasmid was named PUG6-XYL2-YQAB. PCR amplification was performed using the PUG6-XYL2-YQAB plasmid as a template and xyl2up-s and xyl2 down-a as primers to obtain KL-Δ XYL::EcYQAB-loxp-kan-loxp Expression cassette or KL-ΔXYL2::DdYQAB-loxp-kan-loxp Expression cassette.

[0160] 8. WRONG Construction of mutants

[0161] Using a point mutation kit, the catalytic active site of KL-GalT was mutated to construct KL-GALT M1 , KL-GALT M2 and KL-GALT M3 The three strains had the amino acid residues of C54A / N166A / H179A / H294A / Q338A, C51A / H121A / 195EA / H313A / E332A or H177A / H311A / K326A / F327A / V329A mutated based on SEQ ID NO:5.

[0162] 9. Construction and verification of genetically engineered strains of Kluyveromyces lactis

[0163] Respectively KL-ΔGALT::loxp-Kan-loxp , KL-ΔGALT::LNBP1-loxp-Kan-loxp The expression cassette was transformed into Kluyveromyces lactis and the G418 resistance was deleted to obtain strains KL-LNB-0 and Strain KL-LNB-1. KL-Δ XYL1::LNBP1-loxp-Kan-loxp The expression cassette was transformed into StrainKL-LNB-1 and the G418 resistance was deleted to obtain strain StrainKL-LNB-2. KL-ΔXYL3::GALK2-loxp-Kan-loxp The expression cassette was transformed into StrainKL-LNB-2 and the G418 resistance was deleted to obtain strain StrainKL-LNB-3. KL-ΔXYL2::EcYAQB-loxp-kan-loxp Expression cassette and KL-ΔXYL2::DdYQAB-loxp-kan-loxp The expression cassette was transformed into StrainKL-LNB-3 and the G418 resistance was deleted to obtain strains StrainKL-LNB-4 and StrainKL-LNB-5. KL-ΔXYL1::LNBP1-loxp-Kan-loxp Expression cassette, KL-ΔXYL3::GALK2-loxp-Kan-loxp Expression cassette and KL-ΔXYL2::EcYQAB-loxp-kan-loxp The expression cassettes were sequentially introduced into the starting Kluyveromyces lactis and then introduced into KL-GALT M1 , KL-GALT M2 , KL-GALT M3 , and obtained strains StrainKL-LNB-6, StrainKL-LNB-7, and StrainKL-LNB-8. KL-ΔXYL1::LNBP1-loxp-Kan-loxp Expression cassette and KL-ΔXYL2::EcYQAB-loxp-kan-loxp Lactobacillus lactis was introduced and G418 resistance was deleted to obtain strain KL-LNB-9. KL-ΔXYL1::LNBP1-loxp-Kan-loxp expression cassette and KL-ΔXYL2:: DdYQAB-loxp-kan-loxp The Kluyveromyces lactis was introduced sequentially and the G418 resistance was deleted to obtain the strain StrainKL-LNB-10.

[0164] Will KL-ΔGALT::LNBP2-loxp-Kan-loxp The expression cassette was transformed into Kluyveromyces lactis and the G418 resistance was deleted to obtain strain StrainKL-LNB-1-1. KL-ΔXYL1::LNBP2-loxp-Kan-loxp The expression cassette was transformed into StrainKL-LNB-1-1 and the G418 resistance was deleted to obtain strain StrainKL-LNB-2-1. KL-ΔXYL3::GALK2- loxp-Can-loxp The expression cassette was transformed into StrainKL-LNB-2 and the G418 resistance was deleted to obtain strain StrainKL-LNB-3-1. KL-ΔXYL2::EcYQAB-loxp-kan-loxpThe Kluyveromyces lactis strain KL-LNB-3-1 was introduced and the G418 resistance was deleted to obtain the strain Strain KL-LNB-4-1. KL-ΔXYL2::DdYQAB-loxp-kan-loxp The lactic acid Kluyveromyces strain KL-LNB-3-1 was introduced and the G418 resistance was deleted to obtain the strain Strain KL-LNB-5-1. KL-ΔXYL2:: EcYQAB-loxp-kan-loxp Expression cassette and KL-ΔXYL2::DdYQAB-loxp-kan-loxp Lactate was transformed into the starting strain and G418 resistance was deleted to obtain strains StrainKL-LNB-6-1 and StrainKL-LNB-7-1. KL-Δ XYL1::LNBP2-loxp-Kan-loxp Expression cassette, KL-ΔXYL3::GALK2-loxp-Kan-loxp Expression cassette and KL-Δ XYL2::EcYAQB-loxp-kan-loxp The expression cassettes were sequentially introduced into Kluyveromyces lactis and KL-GalT M1 , KL-GalT M2 , KL-GalT M3 , and obtained strains StrainKL-LNB-8-1, StrainKL-LNB-9-1, and StrainKL-LNB-10-1. KL-ΔXYL2::EcYQAB-loxp-kan-loxp Expression cassette and KL-ΔXYL2::DdYQAB-loxp-kan- loxp The lactic acid Kluyveromyces strain KL-LNB-0 was introduced and the G418 resistance was deleted to obtain strains Strain KL-LNB-11 and Strain KL-LNB-12.

[0165] The correspondence between the genetically engineered strains and their genotypes is shown in Table 1. To verify the correctness of the above strains, we extracted the genomes of the above transformants and original strains, and amplified the genomes by PCR using the corresponding knockout cassette or expression cassette amplification primers. If the size of the only band obtained after PCR amplification is consistent with the knockout cassette or expression cassette, the strain is judged to be correct, otherwise the strain is judged to be a false positive error.

[0166] Table 1. Genetically engineered strains of Kluyveromyces lactis and their genotypes

[0167] .

[0168] Example 2. Preparation of genetically engineered Kluyveromyces marxianus

[0169] 1. Yeast marker genes KM-ΔGALT::loxp-Kan-loxp Construction of knockout cassette

[0170] The Kluyveromyces marxianus genome was used as a template, km-galt-upF: 5′-aggtgacactataCAGTGTTCCGATCCTTGGTC-3′;

[0171] The upstream and downstream sequences of GALT were amplified by PCR using the primers km-galt-upR: 5'-gtcgacctgGGCTATACTATGCTATGCAATG-3'; km-galt-doF: 5'-tccactagtgCCAGCTTTCTAAAGATGCCA-3'; km-galt-doR: 5'-ctatagggagaCTTTATCATCATGCAGGTGAT-3'. The resistance sequence Kan was amplified by PCR using the pUG6 plasmid containing Kan resistance as a template and the primers km-galt-loxp-F: 5'-CATAGTATAGCCcaggtcgacaacccttaata-3' and km-galt-loxp-R: 5'-AGAAAGCTGGcactagtggatctgatatcacc-3'. Using the PUG6 plasmid as a template, PUG6-km-galt-F: 5'-GCATGATGATAAAGtctccctatagtgagtcgtatt-3' and PUG6-km-galt-R: 5'-TCGGAACACTGtatagtgtcacctaaatcgtat-3' as primers, PCR amplification was performed to obtain the PUG6 plasmid backbone. After PCR amplification, agarose gel electrophoresis was performed, and the target bands of the expected size were recovered and connected with 2* MultiF Seamless Assembly Mix at 50°C for 30 minutes before transformation into Escherichia coli. Positive Escherichia coli transformants were picked for plasmid extraction, and the plasmid was named PUG6-km-GALT. Using this plasmid as a template, km-galt-upF and km-galt-doR as primers, PCR amplification was performed to obtain KM-ΔGALT::loxp-Kan-loxp Knockout box.

[0172] 2. KM-ΔGALT::LNBP1-loxp-Kan-loxp Expression cassette construction

[0173] According to the plasmid information of PUG6-km-GALT and PUG6-GALT-LNBP1, the amplification primers were designed as follows:

[0174] km-galt-lnbp1-F: 5'-TAGCCAAGAACcatatgtatcacgttgacttg-3';

[0175] km-galt-lnbp1-R: 5'-gtcgacctgggaatattacgaggatatgatgc-3';

[0176] km-galt-lnbp1-PUG-F:5'-cgtaatattcccaggtcgacaacccttaata-3';

[0177] km-galt-lnbp1-PUG6-R:5'-gtgatacatatgGTTCTTGGCTATACTATGCTAT-3'.

[0178] Using the PUG6-GALT-LNBP1 plasmid sequence as a template, km-galt-lnbp1-F and km-galt-lnbp1-R as primers, PCR amplification was performed to obtain the lnbp1 expression cassette sequence. Using the PUG6-km-GALT plasmid sequence as a template, km-galt-lnbp1-PUG-F and km-galt-lnbp1-PUG6-R as primers, PCR amplification was performed to obtain the lnbp1 expression cassette sequence. WRONG Upstream and downstream homology arms, Kan resistance sequence and PUG6 backbone sequence. Agarose gel electrophoresis recovery LNBP1 Expression cassette sequence and WRONG The upstream and downstream homology arms-Kan resistance sequence-PUG6 backbone sequence bands were connected and then transformed into E. coli. The positive transformants were selected to extract the plasmid and named PUG6-km-GALT-LNBP1. PUG6-km-GALT-LNBP1 was used as a template and km-galt-upF and km-galt-doR were used as primers for PCR amplification to obtain KM-ΔGALT::LNBP1-loxp-Kan-loxp Expression cassette.

[0179] 3. KM-ΔGALT::LNBP2-loxp-Kan-loxp Expression cassette construction

[0180] According to the plasmid information of PUG6-km-GALT-LNBP1 and PUG6-XYL1-LNBP2, the amplification primers were designed as follows:

[0181] km-galt-lnbp2-F: 5'-ctgaaagatATGAAGAAGAAGAAGGGTAGAGT-3';

[0182] km-galt-lnbp2-R: 5'-ActggttacaacaTTAACCTTCATTAGAAACATCTTCCCA-3';

[0183] km-galt-lnbp2-PUG-F:5'-CTAATGAAGGTTAAtgttgtaaccagtgtgcgc-3';

[0184] km-galt-lnbp2-PUG6-R:5'-ACCCTTCTTCTTCTTCATctttcagttctcgatgagt-3'.

[0185] Using the PUG6-XYL1-LNBP2 plasmid sequence as a template, km-galt-lnbp2-F and km-galt-lnbp2-R as primers, PCR amplification was performed to obtain the lnbp2 expression cassette sequence. Using the PUG6-km-GALT-LNBP1 plasmid sequence as a template, km-galt-lnbp2-PUG-F and km-galt-lnbp2-PUG6-R as primers, PCR amplification was performed to obtain the lnbp2 expression cassette sequence. WRONG Upstream and downstream homology arms, Kan resistance sequence, LNBP2 Promoter and terminator sequences and PUG6 backbone sequence. Recover each target band by agarose gel electrophoresis. After ligation, transform into E. coli. Take the positive transformant to extract the plasmid, named PUG6-km-GALT-LNBP2. Use it as a template, km-galt-upF and km-galt-doR as primers for PCR amplification to obtain KM-ΔGALT::LNBP2-loxp-Kan- loxp Expression cassette.

[0186] 4. KM-ΔXYL1::LNBP1-loxp-Kan-loxp Expression cassette construction

[0187] According to the upstream and downstream sequences of km-XYL1, PUG6-km-GALT-LNBP1 plasmid, screening resistance Kan and LNBP1 Sequence information of expression cassette, primer design:

[0188] km-xyl1-upF:5'-taggtgacactataATATGGGCAGTGTGATCTGT-3';

[0189] km-xyl1-upR:5'-cgtgatacatatgGGTGGAGATAATTGACACT-3';

[0190] km-xyl1-doF:5'-ccactagtgCATGTCAAATGATGAAACGA-3';

[0191] km-xyl1-doR:5'-actataggggagaGAACCAATAATTGTTACCTTGT-3';

[0192] km-xyl1-lnbp1-kan-F:5'-ATCTCCACCcatatgtatcacgttgacttgg-3';

[0193] km-xyl1-lnbp1-kan-R:5'-ATTTGACATGcactagtggatctgatatcac-3';

[0194] km-xyl1-lnbp1-PUG-F:5'-TTATTGGTTCtctccctatagtgagtcgt-3';

[0195] km-xyl1-lnbp1-PUG6-R: 5'-CTGCCCATATtatagtgtcacctaaatcgtatg-3'.

[0196] Using the Kluyveromyces marxianus genome as a template and km-xyl1-upF, km-xyl1-upR; km-xyl1-doF, km-xyl1-doR as primers, PCR amplification was performed to obtain the upstream and downstream sequences of km-XYL1. LNBP1 The expression cassette and Kan-resistant PUG6-km-GALT-LNBP1 plasmid were used as templates, km-xyl1-lnbp1-kan-F and km-xyl1-lnbp1-kan-R were used as primers, and PCR amplification was performed to obtain the resistance sequences Kan and LNBP1 . Using PUG6-km-GALT-LNBP1 plasmid as template, km-xyl1-lnbp1-PUG-F and km-xyl1-lnbp1-PUG6-R as primers, PCR amplification was performed to obtain the PUG6 plasmid backbone. Agarose gel electrophoresis was used for gel recovery, ligation, transformation, and plasmid extraction, and the plasmid was named PUG6-km-XYL1-LNBP1. Using PUG6-km-XYL1-LNBP1 plasmid as template, km-xyl1-upF and km-xyl1-doR as primers, PCR amplification was performed to obtain KM-ΔXYL1::LNBP1- loxp-Can-loxp Expression cassette.

[0197] 5. KM-ΔXYL1::LNBP2-loxp-Kan-loxp Expression cassette construction

[0198] Using PUG6-XYL1-LNBP2 as a template, km-xyl1-lnbp2-F and km-xyl1-lnbp2-R as primers, the expression cassette sequence of lnbp2 was synthesized; using PUG6-km-XYL1-LNBP1 plasmid as a template, pug6-km-lnbp2-F and pug6-km-lnbp2-R as primers, the backbone-upstream and downstream homology arm km-xyl1 sequence of PUG6 was amplified. After the target band was recovered by agarose gel electrophoresis, it was connected and transformed into Escherichia coli. The extracted plasmid was named PUG6-km-XYL1-LNBP2. Using it as a template, km-xyl1-upF and km-xyl1-doR as primers, PCR amplification was performed to obtain KM-ΔXYL1::LNBP2-loxp-Kan-loxp Expression cassette. The primer sequences are:

[0199] km-xyl1-lnbp2-F: 5'-taggtgacactataATATGGGCAGTGTGATCTGT-3';

[0200] km-xyl1-lnbp2-R:5'-cgtgatacatatgGGTGGAGATAATTGACACT-3';

[0201] pug6-km-lnbp2-F:5'-ccactagtgCATGTCAAATGATGAAACGA-3';

[0202] pug6-km-lnbp2-R: 5'-actataggggagaGAACCAATAATTGTTACCTTGT-3'.

[0203] 6. Construction of KM-ΔXKS1::GALK2-loxp-Kan-loxp expression cassette

[0204] according to XKS1 Upstream and downstream sequences, sequence information of PUG6-XYL3-GALK2 plasmid, primer design:

[0205] km-xks1-upF: 5'-gacactataATATACCGAGAGAATCTCGA-3';

[0206] km-xks1-upR: 5'-aattcgattgagTGCTGAAATATTATGAGAGCG-3';

[0207] km-xks1-doF:5'-ccactagtggCAACCAGAAGCTGTTGAC-3';

[0208] km-xks1-doR:5'-ctcactataggTTGAGCCTCAACATCAATCG-3';

[0209] km-galk-F: 5'-TATTTCAGCActcaatcgaattacacagaaca-3';

[0210] km-galk-R: 5'-TTCTGGTTGccactagtggatctgatatcac-3';

[0211] km-xks1-PUG6-F: 5'-GAGGCTCAAcctatagtgagtcgtattaatttcg-3';

[0212] km-galk-PUG6-R: 5'-TTCTCTCGGTATATtatagtgtcacctaaatcgtatg-3'.

[0213] The Kluyveromyces marxianus genome was used as a template and km-xks1-upF, km-xks1-upR, km-xks1-doF, km-xks1-doR were used as primers to obtain the XKS1 Upstream and downstream sequences. Using the PUG6-XYL3-GALK2 plasmid as a template, km-galk-F and km-galk-R as primers, PCR amplification was performed to obtain the sequence of the resistance sequence Galk-Kan. Using the PUG6-XYL3-GALK2 plasmid as a template, km-xks1-PUG6-F and km-galk-PUG6-R as primers, PCR amplification was performed to obtain the PUG6 plasmid backbone. The plasmid in the positive transformant was obtained by the same method as above and named PUG6-km-XKS1-GALK2. Using this plasmid as a template, km-xks1-upF and km-xks1-doR as primers, amplification was performed to obtain KM-ΔXKS1::GALK2-loxp-Kan-loxp Expression cassette.

[0214] 7. KM-ΔPHA2::YQAB-loxp-kan-loxp Expression cassette construction

[0215] According to the upstream and downstream sequences of Pha2 and the sequence information of PUG6-Xyl2-Yqab plasmid, amplification primers were designed:

[0216] km-pha2-upF: 5'-ggtgacactataATTCCATTTGGATTTGGCAG-3';

[0217] km-pha2-upR: 5'-gtaattcgattgagAGCTACTTTTAGTTGGAGATT-3';

[0218] km-pha2-doF:5'-ccactagtggTTGTTGTTACTTACCATTCCG-3';

[0219] km-pha2-doR:5'-cactataggCAATATGGCTACAGATATAGACC-3';

[0220] km-yqab-kan-F:5'-AAAGTAGCTctcaatcgaattacacagaacac-3';

[0221] km-yqab-kan-R:5'-GTAACAACAAccactagtggatctgatatcac-3';

[0222] km-yqab-PUG6-F:5'-GTAGCCATATTGcctatagtgagtcgtattaatttcg-3';

[0223] km-pha2-yqab-PUG6-R: 5'-CCAAATGGAATtatagtgtcacctaaatcgtatg-3'.

[0224] The Kluyveromyces marxianus genome was used as a template and km-pha2-upF, km-pha2-upR; km-pha2-doF) and km-pha2-doR were used as primers to obtain the PHA2 Upstream and downstream sequences. Using the PUG6-XYL2-YQAB plasmid containing Kan resistance as a template, km-yqab-kan-F and km-yqab-kan-R as primers, PCR amplification was performed to obtain the resistance sequence Kan-Yqab common sequence. Using the PUG6-XYL2-YQAB plasmid as a template, km-yqab-PUG6-F and km-pha2-yqab-PUG6-R as primers, PCR amplification was performed to obtain the PUG6 plasmid backbone. Then, agarose gel electrophoresis, Escherichia coli transformation, and plasmid extraction were performed, and the plasmid was named PUG6-km-PHA2-YQAB. Using the PUG6-km-PHA2-YQAB plasmid as a template, km-pha2-upF and km-pha2-doR) as primers, PCR amplification was performed to obtain KM-ΔPHA2::EcYQAB-loxp-kan-loxp Expression cassette or KM-ΔPHA2:: DdYQAB-loxp-kan-loxp Expression cassette.

[0225] 8. WRONG Construction of mutants

[0226] Using a point mutation kit, the catalytic active site of KM-GALT was mutated to construct KL-GALT M1 , KL-GALT M2 and KL-GALT M3 The three strains had the amino acid residues of C54A / N166A / H179A / H294A / Q338A, C51A / H121A / 195EA / H313A / E332A or H177A / H311A / K326A / F327A / V329A mutated based on SEQ ID NO:6.

[0227] 9. Construction and verification of LNB synthetic strains

[0228] Respectively KM-ΔGALT::loxp-Kan-loxp Knockout box, KM-ΔGALT::LNBP1-loxp-Kan-loxp The expression cassette was transformed into Kluyveromyces marxianus and the G418 resistance was deleted to obtain strains StrainKM-LNB-0 and StrainKM-LNB-1. The G418 resistance in the KM-LNB-1 strain was deleted. KM-ΔXYL1::LNBP1-loxp-Kan-loxp The expression cassette was transformed into StrainKM-LNB-1 and the G418 resistance was deleted to obtain strain StrainKM-LNB-2. KM-ΔXKS1::GALK2- loxp-Can-loxp The expression cassette was transformed into StrainKM-LNB-2 and the G418 resistance was deleted to obtain strain StrainKM-LNB-3. KM-ΔPHA2::EcYQAB-loxp-kan-loxp Expression cassette and KM--ΔPHA2::DdYQAB-loxp-can-loxp The expression cassette was transformed into Strain KM-LNB-3 and the G418 resistance was deleted to obtain strains Strain KM-LNB-4 and Strain KM-LNB-5. The KM-ΔXYL1::LNBP1-loxp-Kan-loxp expression cassette, KM-ΔXKS1::GALK2-loxp-Kan-loxp expression cassette and KM-ΔPHA2::EcYQAB-loxp-kan-loxp expression cassette were sequentially introduced into Kluyveromyces marxianus and respectively. KM-WRONG M1 , KM-WRONG M2 , KM-WRONG M3 , and obtained strains StrainKM-LNB-6, StrainKM-LNB-7, and StrainKM-LNB-8. KM-ΔXYL1::LNBP1-loxp-Kan-loxp Expression cassette and KM-ΔPHA2::EcYQAB- loxp-can-loxp The strain Kluyveromyces marxianus was introduced and the G418 resistance was deleted to obtain the strain StrainKM-LNB-9. KM-ΔXYL1::LNBP1-loxp-Kan-loxp Expression cassette and KM-ΔPHA2::DdYQAB-loxp-kan-loxpThe strain Kluyveromyces marxianus was introduced sequentially and the G418 resistance was deleted to obtain the strain StrainKM-LNB-10.

[0229] Will KM-ΔGALT::LNBP2-loxp-Kan-loxp The expression cassette was transformed into Kluyveromyces marxianus and the G418 resistance was deleted to obtain strain StrainKM-LNB-1-1. KM-ΔXYL1::LNBP2-loxp-Kan-loxp The expression cassette was transformed into StrainKM-LNB-1-1 and the G418 resistance was deleted to obtain strain StrainKM-LNB-2-1. KM-ΔXKS1::GALK2- loxp-Can-loxp The expression cassette was transformed into StrainKM-LNB-2 and the G418 resistance was deleted to obtain strain StrainKM-LNB-3-1.

[0230] Will KM-ΔPHA2::EcYQAB-loxp-kan-loxp The strain KM-LNB-3-1 was introduced into Kluyveromyces marxianus and the G418 resistance was deleted to obtain the strain StrainKM-LNB-4-1. KM-ΔPHA2::DdYQAB-loxp-kan-loxp The strain Kluyveromyces marxianus strain KM-LNB-3-1 was introduced and the G418 resistance was deleted to obtain the strain Strain KM-LNB-5-1. KM- ΔXYL1::LNBP2-loxp-Kan-loxp Expression cassette and KM-ΔPHA2::EcYQAB-loxp-kan-loxp The expression cassette was introduced into the starting strain of Kluyveromyces marxianus and the G418 resistance was deleted to obtain the strain StrainKM-LNB-6-1; KM-ΔXYL1:: LNBP2-loxp-Kan-loxp Expression cassette and KM-ΔPHA2::DdYQAB-loxp-kan-loxp The expression cassette was introduced into the starting strain of Kluyveromyces marxianus to obtain strain Strain KM-LNB-7-1; KM-ΔXYL1::LNBP2-loxp-Kan-loxp Expression cassette, KM-ΔXKS1::GALK2-loxp-Kan-loxp Expression cassette and KM-ΔPHA2::EcYQAB-loxp-kan-loxp The expression cassettes were sequentially introduced into Kluyveromyces marxianus and then into KM-WRONG M1 , KM-WRONG M2 , KM-WRONG M3 , and obtained strains StrainKM-LNB-8-1, StrainKM-LNB-9-1, and StrainKM-LNB-10-1. KM-ΔPHA2::EcYQAB- loxp-can-loxp Expression cassette and KM-ΔPHA2::DdYQAB-loxp-kan-loxp The strains StrainKM-LNB-0 were introduced into Kluyveromyces marxianus and the G418 resistance was deleted to obtain strains StrainKM-LNB-11 and StrainKM-LNB-12.

[0231] The genetically engineered strains and strain genotypes are detailed in Table 2. To verify the correctness of the above strains, we extracted the genomes of the above transformants and original strains, and amplified the genomes by PCR using the corresponding knockout cassette or expression cassette amplification primers. After PCR amplification, if there is a unique band with the same size as the knockout cassette or expression cassette, the strain is judged to be correct, otherwise the strain is judged to be a false positive error.

[0232] Table 2. Recombinant strains of K. marxianus and their genotypes

[0233]

[0234] Example 3. Fermentation synthesis of LNB by genetically engineered Kluyveromyces lactis and Kluyveromyces marxianus

[0235] Lactose or glucose and galactose were used as substrates, and a two-stage culture was used to conduct a LNB synthesis verification experiment using Kluyveromyces lactis cells, Kluyveromyces marxianus cells, the Kluyveromyces lactis genetically engineered bacteria obtained in Example 1, and the Marxianus genetically engineered bacteria obtained in Example 2, respectively.

[0236] 1. Fermentation experiment:

[0237] Phase 1: Bacterial growth phase: Glucose is used as a carbon source to culture yeast, allowing the yeast to grow rapidly until the yeast enters the late logarithmic phase or stable phase. The strain is streaked and cultured in solid culture media such as YDP. After culturing at 30°C for 2-3 days, a single colony is picked and inoculated into 1.5mL YPD liquid culture medium, and cultured overnight at 30°C and 200 rpm. Subsequently, 2% of the inoculum is inoculated into a 50mL liquid culture medium shake flask, and cultured at 30°C and 200 rpm until the OD 600 =1, inoculated at a 2% inoculum into 5 L of YPD medium (yeast extract powder 10 g / L, peptone 20 g / L, glucose 20 g / L) (10 L fermenter), and cultured at 30 °C and 200 rpm for bacterial cell accumulation.

[0238] Phase II: Product synthesis period: 30°C, 200 rpm shaking culture for 40 hours, start to add 20g / L lactose or use 10g / L glucose and 10g / L galactose instead of 20g / L lactose, the total fermentation time is 72 hours. After 72 hours of fermentation, centrifuge the fermentation liquid, collect the supernatant and precipitate respectively, use a high-pressure homogenizer to break the precipitate, boil and centrifuge to remove protein, and combine the supernatant to obtain the final fermentation product of yeast.

[0239] 2. Product Identification

[0240] (1) The standard product and fermentation broth were tested according to the above HPLC analysis method. The HPLC analysis results showed that the peak time rt of the LNB standard product was 10.5 min.

[0241] The fermentation broth of Kluyveromyces lactis obtained in Example 1 and the fermentation broth of Kluyveromyces marxianus obtained in Example 2 both showed strong absorption peaks at around 10.5 min, which was consistent with the peak time of the LNB standard, preliminarily indicating that the yeast genetically engineered bacteria fermented to produce lactose-N-disaccharide (LNB).

[0242] (2) LC-MS analysis

[0243] The product with rt=10.5 min in the HPLC spectrum was subjected to LC-MS analysis. The specific conditions for LC-MS analysis were: AminexHPX-87H (Bio-Rad, Inc., Hercules.), detector: UV detector (Hitachi Chromaster), detection wavelength: 210nm, injection volume: 10μL, flow rate: 0.5ml / min, column temperature: 45℃, mobile phase: 5 mM sulfuric acid aqueous solution; H-ESI mode, molecular weight scanning range 400~900.

[0244] The LC-MS spectrum shows that the molecular weight of the LNB standard product, 384.1459, should be LNB+H; the molecular weight of 406.1315 should be LNB+Na. The mass spectrometry results can be retrieved to obtain the following results: Compound CID: 440994, Chemical Formula: C 14 H 25 NO 11 , Extract Mass: 383.1.

[0245] The LC-MS spectrum of the substance with rt=10.5 min in the HPLC spectrum of the fermentation product is consistent with the LC-MS spectrum of the LNB standard, indicating that the substance with rt=10.5 min in the fermentation broth is LNB.

[0246] In summary, the molecular weight of the product obtained by fermentation of the yeast genetically engineered bacteria of the present invention is about 383, and the peak time is consistent with the peak time of the LNB standard, that is, the fermentation solution obtained by the present invention contains lactose-N-disaccharide (LNB, molecular weight 383.1).

[0247] The LNB yield in the fermentation broth was detected by the above HPLC analysis method, and the results are recorded in Tables 3 and 4 respectively.

[0248] After analysis and determination:

[0249] (1) The Kluyveromyces lactis starting strains, StrainKL-LNB-0, StrainKL-LNB-1, StrainKL-LNB-2, StrainKL-LNB-3, StrainKL-LNB-1-1, StrainKL-LNB-2-1, StrainKL-LNB-3-1, StrainKL-LNB-11, and StrainKL-LNB-12 genetically engineered strains do not produce LNB.

[0250] (2) The Kluyveromyces max starting strains, StrainKM-LNB-0, StrainKM-LNB-1, StrainKM-LNB-2, StrainKM-LNB-3, StrainKM-LNB-1-1, StrainKM-LNB-2-1, StrainKM-LNB-3-1, StrainKM-LNB-11, and StrainKM-LNB-12 genetically engineered strains do not produce LNB.

[0251] (3) The results of LNB production by other genetically engineered strains are recorded in Tables 3 and 4, respectively.

[0252] Table 3. Research on the synthesis of LNB by genetically engineered Kluyveromyces lactis

[0253]

[0254]

[0255] Table 4. Research on LNB production by genetically engineered Kluyveromyces marxianus

[0256] .

[0257] Table 3-4 shows the data in Kluyveromyces lactis or Kluyveromyces marxianus strains:

[0258] (1) Simultaneous introduction of lactose-N-biose phosphorylase ( LNBP ) and fructose-1-phosphate phosphorylase ( YQAB ) can synthesize LNB even when lacto-N-biose phosphorylase from different sources is introduced ( LNBP ) and fructose-1-phosphate phosphorylase from different sources ( YQAB ), can still synthesize LNB;

[0259] (2) Based on (1), by destroying the yeast markers WRONG Gene or modification of the GalT amino acid sequence significantly increased the production of LNB.

[0260] (3) Introducing lactose-N-biose phosphorylase alone ( LNBP ) or fructose-1-phosphate phosphorylase ( YQAB ) or disruption of yeast markers GALT Genetic or modified yeast markers GALT The resulting genetically engineered strain has no ability to synthesize LNB.

[0261] (4) Regardless of whether the carbon source is lactose or a mixture of galactose and glucose, the yield of LNB does not differ much.

[0262] Example 4. Construction of a molecular switch in Kluyveromyces lactis to regulate the production of LNB

[0263] 1. KL-ΔGALE::P tef -CAMR-Tuap-PcamO-AGM1-Tagm-loxp-Kan-loxp Construction of expression cassette:

[0264] Using the Kluyveromyces lactis genome as a template, PCR amplification was performed to obtain the upstream and downstream sequences of GalE. Using the pUG6 plasmid containing Kan resistance as a template, PCR amplification was performed to obtain the resistance sequence Kan. Using the PUG6 plasmid as a template, PCR amplification was performed to obtain the PUG6 plasmid backbone. After PCR amplification, agarose gel electrophoresis was performed. After the target band was recovered, it was connected using 2* MultiF Seamless Assembly Mix. Each fragment was added in equimolar numbers and connected at 50°C for 30 minutes before transformation into Escherichia coli. The positive Escherichia coli transformants were picked for plasmid extraction, and the plasmid was named PUG6-GALE. Using the PUG6-GALE plasmid as a template, PCR amplification was performed to obtain the PUG6-GalE plasmid backbone. Subsequently, the Kluyveromyces lactis genome was used as a template to amplify the promoter tef; the transcription factor synthesized by the gene CAMR As a template, PCR amplification was performed to obtain transcription factors CAMR The sequence is shown in SEQ ID NO.7; the PCR amplification was performed using the Kluyveromyces lactis genome as a template. UAP The CamR responsive promoter sequence synthesized by gene was used as a template, and the promoter CamO was obtained by PCR amplification, as shown in SEQ ID NO.8; the Kluyveromyces lactis genome was used as a template, and the promoter CamO was obtained by PCR amplification. AGM1 The CDS sequence and its termination sequence are shown in SEQ ID NO.9. The PUG6-GALE plasmid backbone, promoter tef, transcription factor CAMR , UAPThe termination sequence, promoter CamO sequence, CDS sequence of AGM1 and its termination sequence bands were recovered and connected for transformation into E. coli. Positive transformants were selected for plasmid extraction and the plasmid was named PUG6-ΔGALE-CAMR-AGM1. PUG6-ΔGALE-CAMR-AGM1 plasmid was used as a template and GalE up-s and GalE down-a were used as primers for PCR amplification to obtain KL-ΔGALE::P tef -CAMR-Tuap-PcamO-AGM1-Tagm- loxp-Kan-loxp Expression cassette. Primers were designed as follows:

[0265] GalE up-s: 5'-ctatagaacgcATGTCTGAAGATAAATACTGTTTGGT-3';

[0266] GalE up-a: 5'-gttgtcgacctgcaCAGATGGATGAGCACCAATAG-3'.

[0267] 2. KL-ΔAGM1::loxp-Kan-loxp Construction of knockout cassette

[0268] according to AGM1 The upstream and downstream sequences of Kluyveromyces lactis were amplified by PCR using the genome of Kluyveromyces lactis as a template. AGM1 The upstream and downstream sequences of Kan resistance were obtained by PCR amplification using the pUG6 plasmid containing Kan resistance as a template. The PUG6 plasmid skeleton was obtained by PCR amplification. The target band was recovered by agarose gel electrophoresis, and E. coli was transformed after ligation. The plasmid was extracted and named PUG6-AGM1. PUG6-AGM1 plasmid was used as a template and AGM1 up-s and AGM1down-a were used as primers for PCR amplification to obtain KL-ΔAGM1::loxp-Kan-loxp Knockout cassette. Primers were designed as follows:

[0269] AGM1 up-s: 5'-cactatagaacgcgttgcgaagtctcatctgtt-3';

[0270] AGM1 up-a: 5'-tgtcgacctgcagtcacggaaagacattggtg-3'.

[0271] 3. Construction and verification of strains regulating LNB production by molecular switches

[0272] Construction of strain KL-LNB-13: KL-ΔAGM1::loxp-Kan-loxp Knockout cassette and KL-Δ GalE::P tef -CAMR-Tuap-PcamO-AGM1-Tagm-loxp-Kan-loxpThe expression cassette was transformed into Kluyveromyces lactis StrainKL-LNB-4 strain, and the G418 resistance was deleted to obtain the StrainKL-LNB-13 strain.

[0273] To verify the correctness of the above strains, we extracted the genomes of the above transformants and wild-type strains, and amplified the genomes by PCR using the corresponding knockout cassette or expression cassette amplification primers. If the size of the only band obtained after PCR amplification is consistent with the knockout cassette or expression cassette, the strain is judged to be correct, otherwise the strain is judged to be a false positive error.

[0274] Example 5. Construction of a molecular switch to regulate LNB production in Kluyveromyces marxianus

[0275] 1. KM-ΔGALE::P tef -CAMR-Tuap-PcamO-AGM1-Tagm-loxp-Kan-loxp Construction of expression cassette

[0276] The Kluyveromyces marxianus genome was used as a template and PCR amplified GALE Upstream and downstream sequences. Using the pUG6 plasmid containing Kan resistance as a template, PCR amplification was performed to obtain the resistance sequence Kan. Using the PUG6 plasmid as a template, PCR amplification was performed to obtain the PUG6 plasmid skeleton. After recovering the target band by agarose gel electrophoresis, it was connected and transformed. The positive Escherichia coli transformants were selected for plasmid extraction, and the plasmid was named PUG6-GALE. Using the PUG6-GalE plasmid as a template, PCR amplification was performed to obtain the PUG6-GALE plasmid skeleton. Subsequently, the promoter tef was amplified using the Kluyveromyces marxianus genome as a template; the transcription factor synthesized by gene CAMR As a template, PCR amplification was performed to obtain transcription factors CAMR Sequence; obtained by PCR amplification using the Kluyveromyces marxianus genome as a template UAP Termination sequence; gene synthesis CAMR The responsive promoter sequence was used as a template to obtain the promoter CamO by PCR amplification; the CDS sequence and the termination sequence of AGM1 were obtained by PCR amplification using the Kluyveromyces marxianus genome as a template. CAMR , the termination sequence of UAP, the promoter CamO sequence, the CDS sequence of AGM1 and its termination sequence bands were recovered and connected using 2* MultiF Seamless Assembly Mix and transformed into E. coli. The positive transformants were selected for plasmid extraction and the plasmid was named PUG6-ΔGALE-CAMR-AGM1. PUG6-ΔGALE-CAMR-AGM1 plasmid was used as a template and GalE up-s and GalE down-a were used as primers for PCR amplification to obtain KM-Δ GALE::P tef -CAMR-Tuap-PcamO-AGM1-Tagm-loxp-Kan-loxp Expression cassette. Primers were designed as follows:

[0277] GalE up-s: 5'-ctatagaacgcATGTCTGAAGATAAATACTGTTTGGT-3';

[0278] GalE up-a: 5'-gttgtcgacctgcaCAGATGGATGAGCACCAATAG-3'.

[0279] 2. KM-ΔAGM1::loxp-Kan-loxp Construction of knockout cassette:

[0280] according to AGM1 The upstream and downstream sequences of Kluyveromyces marxianus were amplified by PCR using the Kluyveromyces marxianus genome as a template. AGM1 The upstream and downstream sequences of Kan resistance were obtained by PCR amplification using the pUG6 plasmid containing Kan resistance as a template. The PUG6 plasmid skeleton was obtained by PCR amplification. The above fragments were subjected to agarose gel electrophoresis, and the target bands were recovered and connected, and then transformed into E. coli. Positive transformants were selected for plasmid extraction, and the plasmid was named PUG6-AGM1. PUG6-AGM1 plasmid was used as a template, and AGM1 up-s and AGM1down-a were used as primers for PCR amplification to obtain KM-ΔAGM1::loxp-Kan-loxp Knockout cassette. Primers were designed as follows:

[0281] AGM1 up-s: 5'-cactatagaacgcgttgcgaagtctcatctgtt-3';

[0282] AGM1 up-a: 5'-tgtcgacctgcagtcacggaaagacattggtg-3'.

[0283] 3. Construction and verification of strains regulating LNB production by molecular switches

[0284] Construction of strain KM-LNB-13: KM-ΔAGM1::loxp-Kan-loxp Knockout cassette and KM-Δ GALE::P tef -CAMR-Tuap-PcamO-AGM1-Tagm-loxp-Kan-loxp The expression cassette was transformed into Kluyveromyces marxianus LNB-4 strain, and the G418 resistance was deleted to obtain the StrainKM-LNB-13 strain.

[0285] To verify the correctness of the above strains, we extracted the genomes of the above transformants and wild-type strains, and amplified the genomes by PCR using the corresponding knockout cassette or expression cassette amplification primers. If the size of the only band obtained after PCR amplification is consistent with the knockout cassette or expression cassette, the strain is judged to be correct, otherwise the strain is judged to be a false positive error.

[0286] Example 6. Fermentation experiment of Kluyveromyces lactis genetically engineered bacteria and Kluyveromyces marxianus genetically engineered bacteria obtained in Examples 4-5

[0287] Using 20 g / L lactose or 10 g / L glucose and 10 g / L galactose instead of lactose as the carbon source and camphor as a molecular switch to regulate small molecules, a two-stage culture was used to conduct a synthesis verification experiment of LNB.

[0288] 1. Fermentation experiment

[0289] The first stage is the bacterial growth stage: the same as the first stage of the fermentation experiment in Example 3.

[0290] The second stage is the product synthesis period: 30℃, 200 rpm shaking culture for 40 hours, start to flow 20 g / L lactose or 10 g / L glucose and 10 g / L galactose, and add 200 µM camphor for fermentation, the total fermentation time is 72 hours. After 72 hours of fermentation, the fermentation liquid is centrifuged, and the supernatant and precipitate are collected separately. The precipitate is crushed by a high-pressure homogenizer, boiled and centrifuged to remove protein, and the supernatant is combined to obtain the final fermentation product of yeast.

[0291] 2. Product identification and content analysis methods are the same as in Example 3. The amount of LNB produced by the yeast genetic engineering strains is recorded in Table 5.

[0292] Table 5. LNB synthesis by genetically engineered strains of Kluyveromyces lactis and Kluyveromyces marxianus

[0293] .

[0294] Data in Table 5 illustrate: Before the introduction of the molecular switch, the metabolic flow inside the yeast was changed by adding small molecules with molecular switch regulation function to the genetically engineered strains of Kluyveromyces lactis and Kluyveromyces marxianus during the stable period after growth, thereby significantly increasing the LNB production by nearly ten times.

[0295] Although the present invention has been described in considerable detail by way of illustration and example for purposes of clear understanding, it will be apparent to one of ordinary skill in the art that any equivalent aspects or modifications may be implemented. Therefore, the present description and examples should not be construed as limiting the scope of the present invention.

Claims

1. A genetically engineered yeast, characterized in that: It contains genes of heterologous lactose-N-disaccharide phosphorylase and fructose-1-phosphate phosphorylase. The genetically engineered yeast is selected from Kluyveromyces lactis and Kluyveromyces marxianus. It can synthesize lactose-N-disaccharide using a culture medium containing lactose or galactose as a raw material. The lactose-N-biose phosphorylase gene is derived from Bifidobacteirum longum, and its amino acid sequence is shown in SEQ ID NO: 1, or derived from Bacillus cereus, and its amino acid sequence is shown in SEQ ID NO:

2. The fructose-1-phosphate phosphorylase gene is derived from Escherichia coli, and its amino acid sequence is shown in SEQ ID NO:3, or is derived from the bacterial sweet potato stem rot fungus Dickeya dadantii, and its amino acid sequence is shown in SEQ ID NO:

4.

2. The genetically engineered yeast according to claim 1, characterized in that: The method also includes the disruption of a yeast marker gene. The amino acid sequence of the marker gene of Kluyveromyces lactis is shown as SEQ ID NO:5, and the amino acid sequence of the marker gene of Kluyveromyces marxianus is shown as SEQ ID NO:6.

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