CN117866789B

CN117866789B - A genetically engineered yeast strain for synthesizing disaccharides

Info

Publication number: CN117866789B
Application number: CN202310009900.1A
Authority: CN
Inventors: 方诩; 李凤仪; 韩丽娟
Original assignee: Shandong Henglu Biotechnology Co ltd
Current assignee: Hengrui Qingdao Biotechnology Co ltd
Priority date: 2023-01-05
Filing date: 2023-01-05
Publication date: 2025-04-29
Anticipated expiration: 2043-01-05
Also published as: CN117866789A; WO2024146465A1

Abstract

The invention belongs to the technical fields of bioengineering, genetic engineering technology and oligosaccharide (rare sugar) synthesis, and particularly relates to a method for producing lactose-N-disaccharide by using genetically modified recombinant saccharomycetes. The technical scheme of the invention is that the recombinant yeast cell is introduced with a recombinant yeast cell which comprises (1) a heterologous milk-N-disaccharide phosphorylase (LNBP) gene and (2) a fructose-1-phosphate phosphorylase (YQAB) gene, and further preferably comprises (3) disruption or modification of a marker gene. The recombinant yeast cells have the property of synthesizing lactose-N-disaccharide (Lacto-N-biose, LNB) from lactose and greatly increase the LNB yield by molecular switching.

Description

Yeast genetic engineering bacteria for synthesizing disaccharide

Technical Field

The invention belongs to the technical fields of bioengineering, genetic engineering technology and oligosaccharide (rare sugar) synthesis, and particularly relates to a method for producing lactose-N-disaccharide by using genetically modified yeast genetic engineering bacteria.

Background

Breast milk is the most ideal natural "functional food" for infants, and is the "gold standard" for infant nutrition. Milk-fed infants have a lower survival rate and a higher infection rate than breast-fed infants, and this phenomenon has led to scientists' interest in the study of the composition of breast milk. Since the 21 st century, researchers have found that the total HMOs (acidic and neutral oligosaccharides) concentration in human colostrum is as high as 24 g/L, but decreases with the prolongation of lactation period, and that HMOs are very important for the growth and development of infants and later health, which promote the intestinal microecological balance of infants, promote the proliferation of beneficial bacteria in the intestinal tract, inhibit the growth of harmful bacteria, resist pathogenic bacterial infection, regulate the immune system, promote the cognitive development of infants, etc., and many beneficial effects of breast milk are attributed to human milk oligosaccharides (human milk oligosaccharides, HMOs).

With the continued advancement of analytical tools and techniques for structural characterization, scientists are working to identify components in breast milk, particularly the abundance of HMOs, more than 200 have now been identified. Among them, 2-fucosyllactose (2' -FL), lacto-N-trisaccharide (Lacto-N-tetriaose II, LNTII), lacto-N-tetrasaccharide (Lacto-N-tetraose, LNT) and lacto-N-neotetraose (Lacto-N-neotetraose, LNnT) were found to be the main components in HMOs, exerting the effect of bifidus factor. The study proves that the infant formula milk powder taking cow milk or sheep milk as the main raw material is added with HMOs, and the infant formula milk powder can be fed to infants, and the infant formula milk powder can be close to the breast milk feeding effect in nutrition, immunity and other aspects of infants. Therefore, the addition of the HMOs with physiological functions to infant milk powder, some artificial milk, dairy products, infant food, functional health care products or special crowd food can greatly improve the physical quality of the corresponding crowd, improve the quality of life of the corresponding crowd, and has obvious social and economic values. Infant formulas and dietary supplements to which HMOs are added have been marketed in the united states, the european union, australia and new zealand. Currently, the European Union EFSA, the United states FDA, has approved the marketing of lactose-N-tetraose (LNT) and lactose-N-neotetraose (LNnT).

Lactose-N-disaccharide (Lacto-N-biose, LNB, galβ1-3GlcNAc, chemical formula 1) is one of HMOs and is also the core structure of neutral HMO, and is an important precursor for synthesizing other neutral HMOs, but because of limited source routes, it is highly desirable to establish a synthetic route for obtaining large quantities of lacto-N-disaccharide (LNB) with a simple, efficient and economical core structure. LNB is a disaccharide in which galactose-1-phosphate and acetylglucosamine (GlcNAc) are linked through a1, 3-beta linkage, and in recent years, researchers have been optimizing the LNB production process. In the existing preparation method, the human breast milk is extracted, and the preparation method has the advantages of higher production cost, low yield and high price. Chemical synthesis, while obtaining structurally defined oligosaccharides, requires the participation of heavy metals, is environmentally unfriendly and has food safety issues that limit the commercial progress of chemical synthesis of LNBs. Compared with chemical methods, biological methods have the advantages of simple process, mild reaction conditions, simple product treatment process and the like, so researchers are trying to synthesize lactose-N-disaccharide (LNB) by using biological methods so as to break through the bottleneck, and the LNB can be produced in a large scale at low cost.

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Nishimoto et al (biosci. Biotech. Bioch. 2007, 71 (8), 2101-2104.) synthesized lactose-N-disaccharide (LNB) by in vitro enzyme catalysis, designed a one pot synthesis of LNB pathway using sucrose and acetamido glucose as substrates, catalyzed by sucrose phosphatase to glucose-1-phosphate, and after UDP-Gal/UDP-Glc cycle to galactose-1-phosphate, and acetamido glucose was enzymatically catalyzed to LNB. CN201911055516.5 utilizes a multi-enzyme catalytic system with good biological safety and wide application, and galactose and acetylglucosamine are introduced into three modules of an ATP regeneration circulating system in the multi-enzyme reaction system, so that compared with the former method, the enzyme synthesis rate is improved by more than 13 times, and the lactose-N-disaccharide synthesis and substrate utilization rate are improved. However, the above methods all have to use acetamido glucose as a substrate, which greatly increases the cost of raw materials for synthesizing LNB.

The synthesis of HMOs by microbial fermentation has been focused on the biosynthesis of HMOs by fermentation using escherichia coli. Several patents (application numbers CN201611147477.8, CN201680052611.8, CN201611147478.2 and CN 201510751641.5) exist for heterologous production of HMOs by using E.coli as host. Compared with escherichia coli, the yeast fermentation production has the advantages of food safety, high robustness and no phage problem, and is one of ideal hosts for large-scale industrial production of HMOs. CN202010187632.9 discloses a saccharomyces cerevisiae engineering strain, wherein the yeast cells can produce 2' -fucosyllactose after genetic modification, and an important reference is provided for industrial production of HMOs by yeasts. However, to date, no report has been made on the synthesis of LNB using E.coli or yeast.

Disclosure of Invention

The invention aims to provide a yeast genetic engineering bacterium capable of producing lactose-N-disaccharide and application of the yeast genetic engineering bacterium in the aspect of synthesizing lactose-N-disaccharide (Lacto-N-biose, LNB).

The invention provides a new lactose-N-disaccharide synthesis way, does not need to take acetylglucosamine as a substrate, lays a foundation for large-scale industrial production of lactose-N-disaccharide, and has important economic value and social benefit. Meanwhile, the synthesis method is efficient, mild, simple, convenient and feasible, low in cost, suitable for industrial production and high in practical application value.

The technical scheme of the invention is as follows:

a genetically engineered yeast strain has the performance of synthesizing lactose-N-disaccharide (Lacto-N-biose, LNB) from lactose.

Preferably, the LNB is synthesized from lactose as a raw material, and optionally from a medium containing lactose or galactose.

The yeast genetically engineered bacterium preferably comprises (1) a heterologous lacto-N-disaccharide phosphorylase (Lnbp) gene and (2) a fructose-1-phosphate phosphorylase (YqaB) gene. The yeast genetically engineered bacterium does not need to introduce special transport protein, and has the property of synthesizing lactose-N-disaccharide (Lacto-N-biose, LNB) by lactose.

The lactose-N-disaccharide phosphorylase (Lacto-N-biose phosphorylase, lnbp), also known as 1, 3-beta-galactoside-N-acetylhexosamine phosphorylase (1, 3-beta-galactosyl-N-acetylhexosamine phosphorylase), EC 2.4.1.21. Comprising an enzyme having an amino acid sequence 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 having activity to produce lactose-N-disaccharide LNB.

The lactose-N-disaccharide phosphorylase Lnbp is preferably LNBP1 (Genbank No. BAD80751.1, PDB:2 ZUS) derived from Bifidobacterium longum Bifidobacteirum longum, the amino acid sequence of which is shown in SEQ ID NO: 1.

The lactose-N-disaccharide phosphorylase Lnbp is preferably LNBP2 (Genbank No. WP_ 078422288.1) derived from Bacillus cereus, and the amino acid sequence of the lactose-N-disaccharide phosphorylase is shown as SEQ ID NO. 2.

As shown in FIG. 1, the amino acid sequence identity of SEQ ID NO. 1 to SEQ ID NO. 2 is 52.88%.

The fructose-1-phosphate phosphorylase (YqaB) comprises an enzyme having an amino acid sequence that is 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%, at least 100% sequence identity to SEQ ID No. 3 or SEQ ID No. 4.

The fructose-1-phosphate phosphorylase (YqaB) is preferably EcYQAB derived from Escherichia coli ESCHERICHIA COLI (Genbank No. APQ 20426.1), and the amino acid sequence of the fructose-1-phosphate phosphorylase is shown as SEQ ID NO: 3.

The fructose-1-phosphate phosphorylase (YqaB) is preferably DdYQAB derived from bacterial sweet potato stem rot germ DICKEYADADANTII (Genbank No. WP_ 013319095.1), and the amino acid sequence of the fructose-1-phosphate phosphorylase is shown as SEQ ID NO. 4.

As shown in FIG. 2, the amino acid sequence identity of SEQ ID NO. 3 to SEQ ID NO.4 is 67%.

In some embodiments, the introduction of heterologous lactose-N-disaccharide phosphorylase genes (LNBP) and fructose-1-phosphate phosphorylase genes (YQAB) into yeast cells as hosts is described to enhance the ability of the original yeast cells to synthesize LNB.

In some embodiments, the yeast genetic engineering bacteria may be a rhodotorula (Issatchenkiasp.), candida (Candida), kluyveromyces (Kluyveromyces sp.), pichia (Pichia), schizosaccharomyces (Schizosaccharomyces sp.), hansenula (Hansenula sp.), torulaspora (Torulaspora sp.), yeast (Yeast sp.), zygosaccharomyces sp.) or Saccharomyces (Saccharomyces sp.) yeast cell.

Further preferably, the yeast genetically engineered bacteria are cells such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), kluyveromyces lactis (Kluyveromyces lactis), kluyveromyces marxianus (Kluyveromycesmarxinus), yarrowia lipolytica (yarrowia lipolytica), rhodotorula graminis (Rhodotorulagraminis) and Saccharomyces pastorianus (Saccharomyces pastorianus), wherein the Kluyveromyces lactis (Kluyveromyces lactis) is a yeast cell passing GRAS food safety certification of the United states food and drug administration, and is more acceptable in terms of food or food additive management.

The yeast strain, in a preferred embodiment, the Saccharomyces cell may be selected from the group consisting of Kluyveromyces lactis (Kluyveromyces lactis) and Kluyveromyces marxianus (Kluyveromycesmarxianus). Kluyveromyces lactis has a deposit number ATCC 8585 in the ATCC deposit center. Kluyveromyces marxianus strain in ATCC the deposit center deposit number ATCC 46537.

The strains of the above species can also be easily obtained to the public in many culture collections in China, such as China general microbiological culture Collection center (CGMCC), china Center for Type Culture Collection (CCTCC), guangdong province microorganism culture collection center (GDMCC), and the like.

In one aspect, the yeast genetically engineered bacterium comprises (1) an introduction comprising a milk-N-disaccharide phosphorylase gene (LNBP), (2) an introduction comprising a fructose-1-phosphate phosphorylase gene (YQAB), and (3) a disruption or modification of a yeast marker gene.

The yeast marker gene is galactose-1-phosphate uridyltransferase Gene (GALT). Disruption or modification of the yeast marker Gene (GALT) is a necessary condition to achieve high LNB yields.

The term "disruption" or "modification" means that the coding region and/or control sequence of the reference gene is modified, either partially or completely (e.g., by deletion, insertion, and/or substitution of one or more nucleotides), such that expression of the encoded polypeptide is absent (inactivated) or reduced, and/or enzymatic activity of the encoded polypeptide is absent or reduced. The destructive effects may be measured using techniques known in the art, such as using cell-free extract measurements from the references herein to detect the absence or decrease in galactose consumption.

In certain embodiments, the yeast genetic engineering bacteria comprise a disruption or modification of one or more yeast marker genes.

The yeast marker gene comprises a partial amino acid sequence of galactose-1-phosphate uridyltransferase (GalT) that is 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% identical to the amino acid sequence of SEQ ID NO 5 or SEQ ID NO 6.

The yeast genetically engineered bacterium, preferably, is a kluyveromyces lactis genetically engineered bacterium, further preferably, comprises at least one yeast marker gene disrupted or modified and contains an amino acid sequence of the yeast marker, and a nucleic acid sequence encoding the yeast marker. The nucleic acid sequence Genebank number X07039.1 of the yeast marker gene and the amino acid sequence Genebank number CAA30091.1 of the yeast marker gene are shown as SEQ ID NO. 5.

The Kluyveromyces marxianus genetically engineered bacterium, further preferably, at least one yeast marker gene is disrupted or modified and includes an amino acid sequence of the yeast marker, and a nucleic acid sequence encoding the yeast marker. The nucleic acid sequence Genebank number NC_036026.1 of the yeast marker gene and the amino acid sequence Genebank number XP_022674659.1 of the yeast marker gene are shown as SEQ ID NO. 6.

In one aspect, is a genetically engineered host cell having an actively synthesized LNB pathway, wherein the cell comprises a disruption or modification of a yeast marker gene GALT (e.g., SEQ ID NO:5 and SEQ ID NO: 6). In some embodiments, the genetically engineered host strain has a greater increase in LNB production when cultured under the same conditions as the parent strain.

As shown in FIG. 3, the amino acid sequence identity of SEQ ID NO. 5 to SEQ ID NO. 6 is 79.84%.

The recombinant construction technology of the Kluyveromyces lactis genetic engineering bacteria or Kluyveromyces marxianus genetic engineering bacteria comprises the following steps:

(1) Construction of an expression cassette, introduction of a lactose-N-disaccharide phosphorylase Gene (LNBP) in a Kluyveromyces lactis cell or Kluyveromyces marxianus cell, and/or

(2) Constructing an expression cassette, introducing a fructose-1-phosphate phosphorylase gene (YQAB) into a Kluyveromyces lactis cell or Kluyveromyces marxianus cell, and/or

(3) A knock-out kit is constructed or a point mutation kit is utilized to knock out or modify a yeast marker Gene (GALT) in a Kluyveromyces lactis cell or a Kluyveromyces marxianus cell.

In some embodiments, the yeast genetically engineered strain modulates a CAMO promoter by introducing an artificial transcription factor CamR.

Preferably, the yeast genetically engineered bacterium increases the yield of LNB by modulating the path of GlcNAc-6-P to GlcNAc-1-P.

Another embodiment of the invention is the use of said yeast genetically engineered bacterium in the synthesis of lactose-N-disaccharide (LNB).

The application of the yeast genetic engineering bacteria in synthesizing lactose-N-disaccharide (LNB) takes lactose or glucose and galactose as substrates.

The application preferably includes the step of generating an LNB.

Preferably, the fermentation product of the yeast genetically engineered bacterium can be purified by centrifugation, filtration, decolorization, nanofiltration, chromatographic purification, crystallization, recrystallization and other modes.

In a further embodiment of the present invention, there is provided lactose-N-disaccharides synthesized according to the above-mentioned technical scheme. In addition, based on the synthesis method of the present invention, it is also within the scope of the present invention to synthesize all oligosaccharides or polysaccharides containing lactose-N-disaccharide as a skeletal structure.

Definition of the definition

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 bacterium" is defined herein as a non-naturally occurring host cell that includes one or more (e.g., two, several) heterologous polynucleotides. The host cell may be a mutant strain described herein, or further disrupted to provide a mutant strain described herein.

Heterologous polynucleotide the term "heterologous polynucleotide" is defined herein as a polynucleotide that is not native to the host cell, a native polynucleotide that has been structurally modified with respect to the coding region, a native polynucleotide whose expression has been quantitatively altered by manipulation of the DNA by recombinant DNA techniques, e.g., a different (exogenous) 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.

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. [ journal of molecular biology ],1970, 48:443-453), as implemented in the Needle program of the EMBOSS package (EMBOSS: the EuropeanMolecular Biology Open Software Suite [ open software package of european molecular biology ], rice et al, TRENDS GENET. [ genetics trend ],2000, 16:276-277), preferably version 3.0.0 or higher.

Advantageous effects

The invention utilizes Kluyveromyces genetic engineering bacteria for the first time, and realizes the high-efficiency synthesis of LNB by utilizing only cheap carbon sources such as lactose without adding coenzyme factors such as ATP and acetylglucosamine (GlcNAc). Is superior to in vitro enzyme catalysis method in food safety, energy utilization and production cost. This is also the first time LNB synthesis achieved by microbial fermentation. The catalytic efficiency and the conversion rate of the product are high, the waste discharge is low, the downstream purification effect of the product is facilitated, and the high-purity LNB product is easier to obtain.

The technology has positive significance for the industrial production of the human milk oligosaccharide LNB, and the LNB obtained by the method is green and safe, can be produced in a large scale efficiently and sustainably, has important practical value and has wide development prospect.

Drawings

FIG. 1 shows the alignment of the amino acid sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2.

FIG. 2 shows the alignment of the amino acid sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4.

FIG. 3 shows the alignment of the amino acid sequences shown in SEQ ID NO:5 and SEQ ID NO: 6.

FIG. 4 is an HPLC plot of StrainKL-LNB-4 fermentation product.

Fig. 5 LC-MS diagram of lnb standard.

Fig. 6 LC-MS plot of products with rt=10.5 min in HPLC plot.

Detailed Description

The present invention will be further described in detail by way of examples, and it should be understood that the embodiments described herein are merely illustrative of the present invention and are not intended to limit the invention, and that modifications and alterations to the details and forms of the present invention may be made without departing from the spirit and scope of the invention.

It is noted that the terminology used is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. Furthermore, it will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, devices, components, and/or groups thereof. It is to be understood that the scope of the invention is not limited to the specific embodiments described below, and that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the invention. Experimental methods in the following embodiments, unless specific conditions are noted, are generally in accordance with conventional methods and conditions of molecular biology within the skill of the art, and are fully explained in the literature. See, for example, sambrook et al, molecular cloning, A laboratory Manual, or according to manufacturer's recommendations.

In the following embodiments, unless otherwise specified, experimental methods for specific conditions are generally followed by methods and conditions conventional in molecular biology within the skill of the art, which are fully explained in the literature, and all materials, reagents, etc., are commercially available unless otherwise specified.

In the following examples, the Kluyveromyces lactis was deposited with ATCC 8585 and Kluyveromyces marxianus was deposited with ATCC46537.

In the following examples, all high fidelity enzymes used in the PCR amplification process were purchased from Northena Biol.Co. MultiF Seamless Assembly Mix was purchased from ABclonalTechnology biosystems, inc.

In the following examples, the PCR amplification system was Phanta Super-FIDELITY DNA Polymerase 1. Mu.L, 2X Phanta MaxBuffer. Mu.L, dNTPs (10 mM) 1. Mu.L, primers F and R each 2. Mu.L, template DNA 1. Mu.L, and ultrapure water was added to a total volume of 50. Mu.L. The PCR amplification procedure was 95℃pre-denatured for 3 min,95℃denatured for 15 s,54℃annealed for 30 s,72℃extended for 2-3 min,2-4 steps cycled 30 times, 72℃final extended for 5 min and finally 4℃stored.

In the following examples, the target gene bands recovered by agarose gel electrophoresis were ligated using 2*MultiF Seamless Assembly Mix in a system of DNA fragment (0.5-1 pmol) X. Mu.L, 2*MultiF Seamless Assembly Mix5. Mu.L, and ultrapure water was added to a total volume of 10. Mu.L.

Preferably, the Kluyveromyces genetic engineering bacteria can transfer lactose into cells and degrade the lactose into galactose and glucose in the cells, glucose is catalyzed by multi-step reactions to generate acetylglucosamine-6-phosphate besides entering glycolysis for yeast growth and energy metabolism, the acetylglucosamine-6-phosphate is catalyzed by fructose-1-phosphate phosphorylase (YqaB) to generate acetylglucosamine, galactose is catalyzed to galactose-1-phosphate, and finally the galactose-1-phosphate and the acetylglucosamine are synthesized into LNB by lactose-N-disaccharide phosphorylase (Lnbp).

In the following examples, the yeast transformation process is specifically as follows:

(1) Firstly, preparing yeast competent cells, namely streaking a small amount of yeast strain frozen stock on a flat solid culture medium, and culturing for 2 days in an inverted way at 30 ℃. Yeast single colonies were picked in 50 mL liquid medium, cultured at 30℃and 220: 220 rpm to an OD ₆₀₀ of between 0.8 and 1.5. The cells were collected, washed with 25 mL sterile water, centrifuged at 1500 Xg at room temperature for 10: 10 min, and the supernatant was discarded. 1 mL of 100 mM lithium chloride buffer was added, the pellet was resuspended, centrifuged at 12000 rpm for 30s, and the supernatant was discarded. Adding 400 mu L of 100 mM lithium chloride buffer again, re-suspending and precipitating to obtain yeast cell competence, and sub-packaging according to 50 mu L/tube for transformation. At the same time, 1 mL salmon sperm DNA 5 min was boiled and rapidly ice-bathed to prepare single-stranded DNA.

(2) Conversion by centrifuging the competent yeast prepared above and removing residual lithium chloride solution with Tips. For each transformation, 50% PEG3350 (240. Mu.L), 1M LiCl (36. Mu.L), 2 mg/mL single-stranded Salmon spilm DNA (25. Mu.L), 5-10. Mu.g/50. Mu. L H ₂ O plasmid DNA (50. Mu.L), vigorous vortex mixing, incubation in a 30 ℃ water bath for 30min until the precipitated cells are completely uniformly distributed, centrifugation in a 42 ℃ water bath heat shock 20~25 min;8000 rpm for 10min, collection of yeast cells, then re-suspension of yeast in 500. Mu.L liquid medium, incubation in a 30 ℃ shaker, and application of 25-100. Mu.L of bacterial liquid to selective medium plates after 1-4 h, and inversion culture at 30 ℃.

In the following example, the yeast genome extraction method comprises the steps of picking single colony of Kluyveromyces into a culture medium of 1 mL YPD (10 mL centrifuge tube), culturing at 30 ℃ and 200 rpm overnight, taking 600 mu L of bacterial liquid in a 1.5ml EP tube, centrifuging the bacterial liquid in 10000 rpm for 1 min, removing supernatant, adding 600-800 mu L of genome extraction buffer and 100 mu L of quartz sand, and fully shaking for 5 min. The water bath 30 min at 65 ℃ was inverted once every 10: 10 min. The supernatant was placed in a fresh 1.5mL EP tube, added with an equal volume of DNA extract, blown and mixed well, and centrifuged at 13000 rpm at 10: 10 min. 400. Mu.L of the supernatant was placed in a fresh 1.5mL EP tube, 0.6 volumes of isopropanol and 40. Mu.L of 3M sodium acetate were added, mixed well, and left to stand at-20℃for 30 min.13000 Centrifugation at 10 min rpm, the supernatant was discarded to obtain the yeast genome. Washed twice with 70% ethanol, dissolved in ddH ₂ O after ethanol has evaporated and stored at-20 ℃.

In the following examples, the fermentation reaction products of the genetically engineered yeast were analyzed by High Performance Liquid Chromatography (HPLC) under the conditions of a chromatographic column type AminexHPX-87H (300X 7.8 mm), a mobile phase of 5mMH ₂SO₄ aqueous solution, a column temperature of 45℃and a sample injection volume of 10. Mu.L, an ultraviolet detector (Hitachi Chromaster), a wavelength of 210 nm and a flow rate of 0.5 mL/min.

The fermentation product of the yeast genetically engineered bacterium can be purified by means of centrifugation, filtration, decolorization, nanofiltration, chromatographic purification, crystallization and the like to obtain a reaction solution or solid with higher purity, and can also be prepared into powder by spray drying or freeze-drying technology.

Example 1 preparation of Kluyveromyces lactis genetically engineered bacterium

1. Construction of Yeast marker Gene KL-delta GALT (loxp-Kan-loxp knockout cassette)

Based on the upstream and downstream sequences of GALT, PUG6 plasmid and sequence information for selection of resistance KAN, amplification primers were designed as shown in table 1. The Kluyveromyces lactis genome is taken as a template, and a primer is designed

galt up-s:5’-cactatagaacgcaaataccattgacatgacagat-3’;

galt up-a:5’-gttgtcgacctgccttgatgaaataaattgggcac-3’;

galt down-s:5’-tccactagtgTGAgcaggcaaagcagaaaata-3’;

galt down-a:5’-cactatagggagagaatttgaattccatttgagaactg-3’,

The primer PCR amplification gave upstream and downstream sequences of GALT.

PCR amplification was performed using pUG6 plasmid containing Kan resistance as a template ,KanMX4-galt-s：5'-atttcatcaaggcaggtcgacaacccttaat-3'、KanMX4-galt-a：5'-tgctttgcctgcTCAcactagtggatctgatatcacc-3' as a primer to obtain a resistance sequence Kan. And (3) taking the PUG6 plasmid as a template ,PUG6-galt-s：5'-tggaattcaaattctctccctatagtgagtcgtatt-3'、PUG6-galt-a：5'-caatggtatttgcgttctatagtgtcacctaaatcg-3' as a primer, and carrying out PCR amplification to obtain the PUG6 plasmid skeleton.

After the amplification, agarose gel electrophoresis was performed, and the bands of the desired size were recovered and then ligated using 2 x MultiF Seamless Assembly Mix, and the fragments were added in equimolar amounts. Coli transformation was performed after 30min of ligation at 50 ℃. The positive E.coli transformants were picked for plasmid extraction and the plasmid was designated as PUG6-GALT. The plasmid is used as a template, GALT up-s and GALT down-a are used as primers, and the KL-delta GALT is obtained by PCR amplification with the same sequence.

2. Construction of KL-delta GALT lnbp-loxp-Kan-loxp expression cassette

Amplification primers were designed based on the LNBP1 sequence, the promoter and terminator sequence information for LNBP1, and the PUG6-GALT plasmid information, as follows:

Lnbp1-s(Lac4Pro):5’-actgaaagatATGACTAGCACCGGCCGCT-3’;

Lnbp1-a:5’-tggttacaacaTCAGGCTTCACGCCAGGCGA-3’;

Lac4Pro-s(lnbp1/2):5’-catcaagcatatgtatcacgttgacttgg-3’;

Lac4Pro-a(lnbp1):5’-CGGTGCTAGTCATatctttcagttctcgatgag-3’;

Ter-lnbp1-s:5’-CGTGAAGCCTGAtgttgtaaccagtgtgcgc-3’;

Ter-lnbp/2-a:5’-cgacctggaaaattattggaatattacgaggat-3’;

PUG6-galt-lnbp1/2-s:5’-attccaataattttccaggtcgacaacccttaata-3’;

PUG6-galt-lnbp1/2-a:5’-gtcaacgtgatacatatgcttgatgaaataaattgggcac-3’。

The LNBP1 coding sequence which is artificially synthesized and subjected to codon optimization is used as a template, lnbp1-s (Lac 4 Pro) and Lnbp-a are used as primers, and the LNBP1 sequence is obtained through PCR amplification. The lactic acid Kluyveromyces genome is used as a template, lac4Pro-s (LNBP/2) and Lac4Pro-a (LNBP) are used as primers, and the LNBP1 promoter is obtained through PCR amplification. The stop sequence of LNBP1 is obtained by PCR amplification by taking the genome of the Kluyveromyces lactis as a template, ter-LNBP1-s and Ter-LNBP/2-a as primers. The PUG6-GALT plasmid skeleton is obtained by PCR amplification with the PUG6-GALT plasmid as template, PUG6-GALT-lnbp1/2-s and PUG6-GALT-lnbp1/2-a as primer. Agarose gel electrophoresis was performed as above, and after recovering the promoter, termination sequence and PUG6-GALT plasmid backbone band of LNBP1, equimolar numbers of each fragment were ligated using 2 x MultiF Seamless Assembly Mix. Coli transformation was performed after 30min of ligation at 50 ℃. The positive E.coli transformant was picked for plasmid extraction and the plasmid was designated PUG6-GALT-LNBP 1. PCR amplification is carried out by taking PUG6-GALT-lnbp1 as a template, GALT up-s and GALT down-a as primers, so that the KL-delta GALT lnbp-loxp-Kan-loxp expression cassette can be obtained.

Construction of KL-DeltaGALT lnbp-loxp-Kan-loxp expression cassette

Designing amplification primers according to LNBP2 sequence, promoter and terminator sequence information of LNBP2 and PUG6-GALT plasmid information, wherein the amplification primers comprise:

Lnbp2-s(Lac4Pro):5’-ctgaaagatATGAAGAAGAAGAAGGGTAGAGT-3’;

Lnbp2-a:5’- ActggttacaacaTTAACCTTCATTAGAAACATCTTCCCA-3’;

Lac4Pro-a(lnbp2):5’-ACCCTTCTTCTTCTTCATatctttcagttctcgatgagt-3’;

Ter-lnbp2-s:5’-CTAATGAAGGTTAAtgttgtaaccagtgtgcgc-3’。

The LNBP2 coding sequence which is synthesized artificially and optimized by codons is used as a template, lnbp2-s (Lac 4 Pro) and Lnbp-a are used as primers, and the LNBP2 sequence is obtained by PCR amplification. The lactic acid Kluyveromyces genome is used as a template, lac4Pro-s (LNBP 1/2) and Lac4Pro-a (LNBP) are used as primers, and the LNBP2 promoter is obtained through PCR amplification. The Kluyveromyces lactis genome is used as a template, ter-lnbp/2-a and Ter-lnbp-s are used as primers, and a termination sequence of lnbp is obtained through PCR amplification. The PUG6-GALT plasmid skeleton is obtained by PCR amplification with the PUG6-GALT plasmid as template, PUG6-GALT-lnbp1/2-s and PUG6-GALT-lnbp1/2-a as primer. Agarose gel electrophoresis was performed in the same manner as above, and the LNBP2 promoter, termination sequence and PUG6-GALT plasmid backbone band were recovered and ligated. And (3) transforming into escherichia coli. The positive E.coli transformants were picked for plasmid extraction and the plasmid was designated PUG6-GALT-LNBP2. And (3) carrying out PCR amplification by taking PUG6-GALT-LNBP2 as a template and taking GALT up-s and GALT down-a as primers to obtain the KL-delta GALT:LNBP2-loxp-Kan-loxp expression cassette.

Construction of KL-DeltaXYL 1:: LNBP1-loxp-Kan-loxp expression cassette

Based on the sequence information of the XYL1 upstream and downstream sequences, the PUG6 plasmid, the screening resistance Kan and the LNBP1 expression cassette, the amplification primers are designed as follows:

xyl1 up-s:5’-gtgacactatagactcgagcttgaaacttcga-3’;

xyl1 up-a:5’-gttgtcgacctgctcaccattattgcggaagag-3’;

xyl1 down-s:5’-gatccactagtgAACTTTTCAGGTGCGTTGAT-3’;

xyl1 down-a:5’-ctatagggagaaggtgagtggcatcaattatg-3’;

KanMX4-xyl1-s :5’-caataatggtgagcaggtcgacaacccttaat-3’;

KanMX4-xyl1-a :5’-acCTGAAAAGTTcactagtggatctgatatcacc-3’;

PUG6-xyl1-s:5’-tgccactcaccttctccctatagtgagtcgta-3’;

PUG6-xyl1-a:5’-caagctcgagtctatagtgtcacctaaatcgtat-3’;

PUG6-xyl1-lnbp*2-s:5’-ttcaagcgggaacagcaggtcgacaacccttaat-3’;

PUG6-xyl1-lnbp*2-a:5’-cgtgatacatatgtcaccattattgcggaagag-3’;

Ter-lnbp*2-s:5’-CGTGAAGCCTGAaaatagtttccatgtagaatttcg-3’;

Ter-lnbp*2-a:5’-acctgctgttcccgcttgaagttagc-3’;

Lnbp*2-s:5’-cgcaataatggtgacatatgtatcacgttgacttgg-3’;

Lnbp*2-a:5’-TGgaaactatttTCAGGCTTCACGCCAGGCGATA-3’。

The method comprises the steps of taking a kluyveromyces lactis genome as a template, and XYL1 up-s and XYL1 up-a, and XYL1 down-s and XYL1 down-a as primers, and carrying out PCR amplification to obtain the upstream and downstream sequences of the XYL1. The pUG6 plasmid containing Kan resistance is used as a template, kanMX4-xyl1-s and KanMX4-xyl1-a are used as primers, and a resistance sequence Kan is obtained through PCR amplification. And taking the PUG6 plasmid as a template, taking the PUG6-xyl1-s and the PUG6-xyl 1-a) as primers, and carrying out PCR amplification to obtain the PUG6 plasmid skeleton. After the target bands obtained by agarose gel electrophoresis were ligated at 50℃for 30min with 2X MultiF Seamless Assembly Mix, E.coli transformation was performed, and the plasmid was extracted from the positive E.coli transformant and designated as PUG6-XYL1. The PUG6-XYL1 plasmid skeleton is obtained by PCR amplification by taking the PUG6-XYL1-lnbp x 2-s and the PUG6-XYL1-lnbp x 2-a as templates and the primers. Then, using the lactic acid Kluyveromyces genome as a template, ter-LNBP x 2-s and Ter-LNBP x 2-a as primers, carrying out PCR amplification to obtain a termination sequence, using the PUG6-GALT-LNBP1 plasmid as a template, lnbp x 2-s and Lnbp x 2-a as primers, and carrying out PCR amplification to obtain LNBP1 and a promoter thereof. The plasmid was named PUG6-XYL1-LNBP1 by recovering, ligating, and transforming E.coli into the backbone of the PUG6-XYL1 plasmid, the LNBP1 and its promoter and termination sequence bands, and was extracted. The KL-delta XYL1 is obtained by PCR amplification by taking the PUG6-XYL1-LNBP1 plasmid as a template and XYL1 up-s and XYL1 down-a as primers.

Construction of KL-DeltaXYL 1:: LNBP2-loxp-Kan-loxp expression cassette

Based on the relevant plasmid information, the corresponding primers were designed as follows:

lnbp2-KL-XYL1-F:5’-gagaactgaaagatATGAAGAAGAAGAAGGGTAGAG-3’;

lnbp2-KL-XYL1-R:5’-atggaaactatttTTAACCTTCATTAGAAACATCTTCCCA-3’;

PUG-lnbp2-XYL1-F:5’-AATGAAGGTTAAaaatagtttccatgtagaatttcg-3’;

PUG6-lnbp2-XYL1- R:5’-CTTCTTCTTCATatctttcagttctcgatgagt-3’。

The sequence of LNBP2 was synthesized using plasmid PUG6-GALT-LNBP2 as template and LNBP-KL-XYL 1-F and LNBP-KL-XYL 1-R as primers. The plasmid skeleton portion was synthesized using the plasmid PUG6-XYL1-LNBP1 as a template and the primers PUG-LNBP2-XYL1-F and PUG-LNBP-XYL 1-R. Agarose gel electrophoresis was then performed to recover the band of expected size and the fragments were ligated using 2 x MultiF Seamless Assembly Mix. After Escherichia coli is transformed, a positive transformant is selected for plasmid extraction, the plasmid is named as PUG6-XYL1-LNBP2, and PCR amplification is carried out by taking the plasmid as a template and XYL1 up-s and XYL1 down-a as primers to obtain the KL-delta XYL 1:LNBP 2-loxp-Kan-loxp expression cassette.

Construction of KL-DeltaXYL 3:: GALK2-loxp-Kan-loxp expression cassette

Based on the sequence information of the XYL3 upstream and downstream sequences, the PUG6 plasmid, the selection resistance Kan and GALK gene, amplification primers were designed as follows:

xyl3 up-s:5’-gacactatagaacgcgtatttgttttggggctaatagt-3’;

xyl3 up-a:5’-gtcgacctgcaccaattctaatctataatttggttca-3’;

xyl3 down-s:5’-tccactagtggGTCGTATTGACAAATGTCAATATG-3’;

xyl3 down-a:5’-cgactcactataggTTAGTGTGATGACGCTTCTCT-3’;

KanMX4-xyl3-s:5’-gattagaattggtgcaggtcgacaacccttaata-3’;

KanMX4-xyl3-a:5’-GTCAATACGACccactagtggatctgatatcac-3’;

PUG6-xyl3-s:5’-CATCACACTAAcctatagtgagtcgtattaatttcg-3’;

PUG6-xyl3-a:5’-acaaatacgcgttctatagtgtcacctaaat-3’;

PUG6-xyl3-galk-s:5’-gaacgtcggtgcaggtcgacaacccttaata-3’;

PUG6-xyl3-galk-a:5’-gtaattcgattgagccaattctaatctataatttggttca-3’;

TefPro-s(galk):5’-agattagaattggctcaatcgaattacacagaaca-3’;

TefPro-a(galk):5’-AGGAACGGACATttttaatgttacttctcttgcagt-3’;

galk-s(TefPro):5’-gaagtaacattaaaaATGTCCGTTCCTATTGTGCCA-3’;

galk-a:5’-gtcgacctgcaccgacgttcattaagaatact-3’。

The method comprises the steps of taking a kluyveromyces lactis genome as a template, and XYL3 up-s and XYL3 up-a, and XYL3 down-s and XYL3 down-a as primers, and carrying out PCR amplification to obtain the upstream and downstream sequences of the XYL3. The pUG6 plasmid containing Kan resistance is used as a template, kanMX4-xyl3-s and KanMX4-xyl3-a are used as primers, and a resistance sequence Kan is obtained through PCR amplification. And (3) taking the PUG6 plasmid as a template, taking PUG6-xyl3-s and PUG6-xyl3-a as primers, and carrying out PCR amplification to obtain the PUG6 plasmid skeleton. All the target bands were ligated and transformed, and positive transformants were picked for plasmid extraction, and the plasmid was designated as PUG6-XYL3. And then taking the PUG6-XYL3 plasmid as a template, taking the PUG6-XYL3-galk-s and the PUG6-XYL3-galk-a as primers, and carrying out PCR amplification to obtain the PUG6-XYL3 plasmid skeleton. Then, using the Kluyveromyces lactis genome as a template, tefPro-s (galk) and TefPro-a (galk) as primers, carrying out PCR amplification to obtain a promoter sequence, and using the Kluyveromyces lactis genome as the template, galk-s (TefPro) and galk-a as primers, carrying out PCR amplification to obtain GALK and a termination sequence thereof. The PUG6-XYL3 plasmid backbone, promoter, GALK and termination sequence bands were ligated and E.coli transformed. Plasmids were extracted from positive transformants and designated as PUG6-Xyl3-GALk. PCR amplification is carried out by taking the PUG6-XYL3-GALK plasmid as a template and XYL3 up-s and XYL3 down-a as primers to obtain the KL-delta XYL3: GALK2-loxp-Kan-loxp expression cassette.

Construction of KL-DeltaXYL 2:: YQAB-loxp-kan-loxp expression cassette

Based on the sequence information of XYL2 upstream and downstream, PUG6 plasmid, screening resistance Kan and YQAB gene, the amplification primer is designed as follows:

xyl2 up-s:5’-cactatagaacgcTCTTcccgtttctccatctgaat-3’;

xyl2 up-a:5’-gacctgcaTGTCTGAACCGCAGATACCG-3’;

xyl2 down-s:5’-cactagtggGATCGGTGCCAAAGAATTAC-3’;

xyl2 down-a:5’-actatagggttacctcttctcgcttcttc-3’;

KanMX4-xyl2-s:5’-CGGTTCAGACAtgcaggtcgacaacccttaat-3’;

KanMX4-xyl2-a:5’-TGGCACCGATCccactagtggatctgatatcac-3’;

PUG6-xyl2-s:5’-gcgagaagaggtaaccctatagtgagtcgtattaatttcg-3’;

PUG6-xyl2-a:5’-acgggAAGAgcgttctatagtgtcaccta-3’;

PUG6-xyl2-yqab-s:5’-attccaataattttctgcaggtcgacaaccct-3’;

PUG6-xyl2-yqab-a:5’-cgattgagGTCTGAACCGCAGATACCG-3’;

TefPro-s(yqab):5’-CTGCGGTTCAGACctcaatcgaattacacagaacac-3’;

TefPro-a(yqab):5’-CGTATCTTTCGTACATttttaatgttacttctcttgcag-3’;

Ter-yqab-s:5’-GTTGCTCGAGtgttgtaaccagtgtgcgct-3’;

Ter-yqab-a:5’-gtcgacctgcagaaaattattggaatattacgaggat-3’;

The method comprises the steps of taking a kluyveromyces lactis genome as a template, and XYL2up-s and XYL2 up-a, and carrying out PCR amplification by taking XYL2 down-s and XYL2 down-a as primers to obtain the upstream and downstream sequences of the XYL 2. The pUG6 plasmid containing Kan resistance is used as a template, kanMX4-xyl2-s and KanMX4-xyl2-a are used as primers, and a resistance sequence Kan is obtained through PCR amplification. And (3) taking the PUG6 plasmid as a template, taking PUG6-xyl2-s and PUG6-xyl2-a as primers, and carrying out PCR amplification to obtain the PUG6 plasmid skeleton. And (3) connecting, converting and extracting plasmids of all target bands, namely the plasmids are named as PUG6-XYL2, taking the plasmids as templates, taking the PUG6-XYL2-yqab-s and the PUG6-XYL2-yqab-a as primers, and carrying out PCR amplification to obtain the PUG6-XYL2 plasmid skeleton. Then, using the Kluyveromyces lactis genome as a template, tefPro-s (yqab) and TefPro-a (yqab) as primers, performing PCR amplification to obtain a promoter sequence, using the Kluyveromyces lactis genome as a template, and using Ter-yqab-s and Ter-yqab-a as primers, performing PCR amplification to obtain a termination sequence. And (3) PCR amplification is carried out by taking the artificially synthesized YQAB coding sequence subjected to codon optimization as a template to obtain EcYQAB or DdYQAB sequence. The PUG6-XYL2 plasmid backbone, promoter, termination sequence and YQAB bands were recovered and ligated for 30min using 2 x MultiF Seamless Assembly Mix for e.coli transformation. The positive E.coli transformants were picked for plasmid extraction and the plasmid was designated PUG6-XYL2-YQAB. PCR amplification is carried out by taking the PUG6-XYL2-YQAB plasmid as a template and XYL2up-s and XYL2 down-a as primers to obtain the KL-DeltaXYL EcYQAB-loxp-kan-loxp expression cassette or KL-DeltaXYL 2 DdYQAB-loxp-kan-loxp expression cassette.

Construction of GALT mutant

The point mutation kit is used for respectively carrying out the mutation of catalytic active sites on KL-GalT to construct three strains KL-GALT ^M1,KL-GALT^M2 and KL-GALT ^M3, and the amino acid residues of C54A/N166A/H179A/H294A/Q338A, C51A/H121A/195EA/H313A/E332A or H177A/H311A/K326A/F327A/V329A are mutated on the basis of SEQ ID NO: 5.

9. Construction and verification of lactic acid Kluyveromyces genetic engineering strain

KL-delta GALT:: loxp-Kan-loxp, KL-delta GALT:: LNBP1-loxp-Kan-loxp expression cassettes are respectively transformed into kluyveromyces lactis and G418 resistance is deleted to obtain strains KL-LNB-0, strainKL-LNB-1 respectively. The KL-DeltaXYL 1:LNBP 1-loxp-Kan-loxp expression cassette was transformed into StrainKL-LNB-1 and G418 resistance was deleted, resulting in strain StrainKL-LNB-2. The KL-DeltaXYL 3:: GALK2-loxp-Kan-loxp expression cassette was transformed into StrainKL-LNB-2 and G418 resistance was deleted, resulting in strain StrainKL-LNB-3. The KL-DeltaXYL 2:: ecYAQB-loxp-kan-loxp expression cassette and the KL-DeltaXYL 2::: ddYQAB-loxp-kan-loxp expression cassette were transformed into StrainKL-LNB-3 and G418 resistance was deleted, respectively, yielding strains StrainKL-LNB-4 and StrainKL-LNB-5. The expression cassettes of KL-DeltaXYL 1, LNBP1-loxp-Kan-loxp, KL-DeltaXYL 3, GALK-loxp-Kan-loxp and KL-DeltaXYL 2, ecYQAB-loxp-Kan-loxp are sequentially introduced into Kluyveromyces lactis and KL-GALT ^M1,KL-GALT^M2,KL-GALT^M3 respectively to obtain strains StrainKL-LNB-6, strainKL-LNB-7 and StrainKL-LNB-8. KL-deltaxyl 1:: LNBP1-loxp-Kan-loxp expression cassette and KL-deltaxyl 2:: ecYQAB-loxp-Kan-loxp are sequentially introduced into kluyveromyces lactis and G418 resistance is deleted, so as to obtain the strain StrainKL-LNB-9. KL-deltaxyl 1:: LNBP1-loxp-Kan-loxp expression cassette and KL-deltaxyl 2:: ddYQAB-loxp-Kan-loxp are sequentially introduced into kluyveromyces lactis and G418 resistance is deleted, so as to obtain the strain StrainKL-LNB-10.

The KL-DeltaGALT:: LNBP2-loxp-Kan-loxp expression cassette was transformed into Kluyveromyces lactis and G418 resistance was deleted to obtain strain StrainKL-LNB-1-1. The KL-DeltaXYL 1:LNBP 2-loxp-Kan-loxp expression cassette was transformed into StrainKL-LNB-1-1 and G418 resistance was deleted to give strain StrainKL-LNB-2-1. The KL-DeltaXYL 3:: GALK2-loxp-Kan-loxp expression cassette was transformed into StrainKL-LNB-2 and G418 resistance was deleted, yielding strain StrainKL-LNB-3-1. KL-DeltaXYL 2:: ecYQAB-loxp-kan-loxp was introduced into Kluyveromyces lactis StrainKL-LNB-3-1 and G418 resistance was deleted to obtain strain StrainKL-LNB-4-1. KL-DeltaXYL 2:: ddYQAB-loxp-kan-loxp was introduced into Kluyveromyces lactis StrainKL-LNB-3-1 and G418 resistance was deleted to obtain strain StrainKL-LNB-5-1. KL-DeltaXYL 2:: ecYQAB-loxp-kan-loxp expression cassette and KL-DeltaXYL 2::: ddYQAB-loxp-kan-loxp are respectively transformed into the Kluyveromyces lactis starting strain and G418 resistance is deleted to obtain strains StrainKL-LNB-6-1 and StrainKL-LNB-7-1 respectively. The KL-DeltaXYL 1:: LNBP2-loxp-Kan-loxp expression cassette, KL-DeltaXYL 3:: GALK2-loxp-Kan-loxp expression cassette and KL-DeltaXYL 2::: ecYAQB-loxp-Kan-loxp expression cassette are sequentially introduced into kluyveromyces lactis, and KL-GalT ^M1,KL-GalT^M2,KL-GalT^M3 are respectively introduced to obtain 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 were introduced into kluyveromyces lactis StrainKL-LNB-0 and G418 resistance was deleted, respectively, to obtain strains StrainKL-LNB-11 and StrainKL-LNB-12.

The corresponding relationship between the genetically engineered strain and its genotype is shown in table 1. To verify the correctness of the above strains, we extracted the genomes of the above transformants and the original strain, and amplified the genomes by PCR using the corresponding knockout or expression cassette amplification primers. And if the size of the unique band obtained after PCR amplification is consistent with that of the knockout box or the expression box, judging that the strain is correct, otherwise, judging that the strain is false positive.

TABLE 1 Kluyveromyces lactis genetic engineering strain and genotype thereof

。

EXAMPLE 2 preparation of Kluyveromyces marxianus genetically engineered bacterium

1. Construction of Yeast marker Gene KM-DeltaGALT:: loxp-Kan-loxp knockout Box

Kluyveromyces marxianus genome is used as a template, and km-galt-upF:5'-aggtgacactataCAGTGTTCCGATCCTTGGTC-3';

km-galt-upR: 5'-gtcgacctgGGCTATACTATGCTATGCAATG-3';km-galt-doF:5'-tccactagtgCCAGCTTTCTAAAGATGCCA-3';km-galt-doR:5'-ctatagggagaCTTTATCATCATGCAGGTGAT-3' As primers, the upstream and downstream sequences of GALT were obtained by PCR amplification. PCR amplification was performed using pUG6 plasmid containing Kan resistance as a template ,km-galt-loxp-F:5'-CATAGTATAGCCcaggtcgacaacccttaata-3'、km-galt-loxp-R:5'-AGAAAGCTGGcactagtggatctgatatcacc-3' as a primer to obtain a resistance sequence Kan. And (3) taking the PUG6 plasmid as a template ,PUG6-km-galt-F:5'-GCATGATGATAAAGtctccctatagtgagtcgtatt-3'、PUG6-km-galt-R:5'-TCGGAACACTGtatagtgtcacctaaatcgtat-3' as a primer, and carrying out PCR amplification to obtain the PUG6 plasmid skeleton. After the PCR amplification, agarose gel electrophoresis is carried out, the band with the size meeting the expected purpose is recovered and then connected for 30min at 50 ℃ by using 2 x MultiF Seamless Assembly Mix, and then the escherichia coli transformation is carried out. The positive E.coli transformants were picked for plasmid extraction and the plasmid was designated as PUG6-km-GALT. The plasmid is used as a template, and KM-GALT-upF and KM-GALT-doR are used as primers for PCR amplification to obtain a KM-delta GALT-loxp-Kan-loxp knockout box.

Construction of the LNBP1-loxp-Kan-loxp expression cassette

The amplification primers were designed based on the plasmid information of PUG6-km-GALT, PUG6-GALT-LNBP1 as follows:

km-galt-lnbp1-F :5’-TAGCCAAGAACcatatgtatcacgttgacttg-3’;

km-galt-lnbp1-R :5’-gtcgacctgggaatattacgaggatatgatgc-3’;

km-galt-lnbp1-PUG-F:5’-cgtaatattcccaggtcgacaacccttaata-3’;

km-galt-lnbp1-PUG6-R:5’-gtgatacatatgGTTCTTGGCTATACTATGCTAT-3’。

The LNBP expression cassette sequence is obtained by PCR amplification using the PUG6-GALT-LNBP1 plasmid sequence as a template and km-GALT-LNBP1-F, km-GALT-LNBP-R as a primer. Taking a PUG6-km-GALT plasmid sequence as a template, km-GALT-lnbp-PUG-F, km-GALT-lnbp-PUG 6-R as a primer, and carrying out PCR amplification to obtain a GALT upstream and downstream homologous arm, a Kan resistance sequence and a PUG6 framework sequence. And (3) recovering the LNBP1 expression cassette sequence and the GALT upstream and downstream homology arm-Kan resistance sequence-PUG 6 framework sequence band by agarose gel electrophoresis, connecting, and then converting into escherichia coli. Positive transformant plasmids were picked and designated as PUG6-km-GALT-LNBP1. And (3) carrying out PCR amplification by taking PUG6-KM-GALT-LNBP1 as a template and KM-GALT-upF and KM-GALT-doR as primers to obtain the KM-delta GALT (LNBP 1-loxp-Kan-loxp) expression cassette.

Construction of the LNBP2-loxp-Kan-loxp expression cassette

Amplification primers were designed based on the plasmid information of PUG6-km-GALT-LNBP1, PUG6-XYL1-LNBP2 as follows:

km-galt-lnbp2-F :5’-ctgaaagatATGAAGAAGAAGAAGGGTAGAGT-3’;

km-galt-lnbp2-R:5’-ActggttacaacaTTAACCTTCATTAGAAACATCTTCCCA-3’;

km-galt-lnbp2-PUG-F:5’-CTAATGAAGGTTAAtgttgtaaccagtgtgcgc-3’;

km-galt-lnbp2-PUG6-R:5’-ACCCTTCTTCTTCTTCATatctttcagttctcgatgagt-3’。

The LNBP expression cassette sequence is obtained by PCR amplification using the PUG6-XYL1-LNBP2 plasmid sequence as a template and km-galt-LNBP2-F, km-galt-LNBP-R as a primer. Taking a PUG6-km-GALT-LNBP1 plasmid sequence as a template, km-GALT-LNBP-PUG-F, km-GALT-LNBP-PUG 6-R as a primer, and carrying out PCR amplification to obtain a GALT upstream and downstream homology arm, a Kan resistance sequence, an LNBP2 promoter and terminator sequence and a PUG6 framework sequence. Each target band was recovered by agarose gel electrophoresis. After ligation, E.coli was transformed. Positive transformant plasmids were taken and designated as PUG6-km-GALT-LNBP2. The KM-delta GALT is taken as a template, KM-GALT-upF and KM-GALT-doR are taken as primers for PCR amplification, and thus the LNBP2-loxp-Kan-loxp expression cassette can be obtained.

Construction of the LNBP1-loxp-Kan-loxp expression cassette

Based on the upstream and downstream sequences of km-XYL1, the PUG6-km-GALT-LNBP1 plasmid, the screening resistance Kan and the sequence information of the LNBP1 expression cassette, primers were designed:

km-xyl1-upF:5’-taggtgacactataATATGGGCAGTGTGATCTGT-3’;

km-xyl1-upR:5’-cgtgatacatatgGGTGGAGATAATTGACACT-3’;

km-xyl1-doF:5’-ccactagtgCATGTCAAATGATGAAACGA-3’;

km-xyl1-doR:5’-actatagggagaGAACCAATAATTGTTACCTTGT-3’;

km-xyl1-lnbp1-kan-F:5’-ATCTCCACCcatatgtatcacgttgacttgg-3’;

km-xyl1-lnbp1-kan-R:5’-ATTTGACATGcactagtggatctgatatcac-3’;

km-xyl1-lnbp1-PUG-F:5’-TTATTGGTTCtctccctatagtgagtcgt-3’;

km-xyl1-lnbp1-PUG6-R:5’-CTGCCCATATtatagtgtcacctaaatcgtatg-3’。

Kluyveromyces marxianus genome is used as a template, km-XYL1-upF and km-XYL1-upR, km-XYL1-doF and km-XYL1-doR are used as primers, and upstream and downstream sequences of km-XYL1 are obtained through PCR amplification. The resistant sequences Kan and LNBP1 were obtained by PCR amplification using a PUG6-km-GALT-LNBP1 plasmid containing an LNBP1 expression cassette and Kan resistance as a template, km-xyl1-LNBP1-Kan-F, km-xyl1-LNBP1-Kan-R as primers. The PUG6 plasmid skeleton is obtained by PCR amplification by taking a PUG6-km-GALT-LNBP1 plasmid as a template and km-xyl1-LNBP1-PUG-F, km-xyl 1-LNBP-PUG 6-R as a primer. The gel was cut for recovery, ligation, transformation, and plasmid extraction using agarose gel electrophoresis, and the plasmid was designated as PUG6-km-XYL1-LNBP1. The plasmid PUG6-KM-XYL1-LNBP1 is used as a template, KM-XYL1-upF and KM-XYL1-doR are used as primers, and the KM-DeltaXYL 1 is obtained by PCR amplification, wherein the LNBP1-loxp-Kan-loxp expression cassette is obtained.

Construction of the LNBP2-loxp-Kan-loxp expression cassette

The method comprises the steps of synthesizing an expression cassette sequence of LNBP2 by taking PUG6-XYL1-LNBP2 as a template, km-Xyl 1-LNBP-F and km-Xyl 1-LNBP-R as primers, and amplifying a skeleton-upstream and downstream homology arm km-XYL1 sequence of PUG6 by taking a PUG6-km-XYL1-LNBP 1 plasmid as the template and taking PUG-km-LNBP-F and PUG 6-km-LNBP-2-R as primers. The agarose gel electrophoresis was used to recover the target band, followed by ligation and E.coli transformation. The extracted plasmid was designated PUG6-km-XYL1-LNBP2. Taking the primer as a template and KM-XYL1-upF and KM-XYL1-doR as primers, and carrying out PCR amplification to obtain the KM-DeltaXYL 1:LNBP 2-loxp-Kan-loxp expression cassette. The primer sequences are as follows:

km-xyl1-lnbp2-F:5’-taggtgacactataATATGGGCAGTGTGATCTGT-3’;

km-xyl1-lnbp2-R:5’-cgtgatacatatgGGTGGAGATAATTGACACT-3’;

pug6-km-lnbp2-F:5’-ccactagtgCATGTCAAATGATGAAACGA-3’;

pug6-km-lnbp2-R:5’-actatagggagaGAACCAATAATTGTTACCTTGT-3’。

6. Construction of KM-DeltaXKS1: GALK2-loxp-Kan-loxp expression cassette

Based on the sequence information of the plasmid PUG6-XYL3-GALK, the primers were designed according to the sequence of XKS1 upstream and downstream:

km-xks1-upF:5’-gacactataATATACCGAGAGAATCTCGA-3’;

km-xks1-upR:5’-aattcgattgagTGCTGAAATATTATGAGAGCG-3’;

km-xks1-doF:5’-ccactagtggCAACCAGAAGCTGTTGAC-3’;

km-xks1-doR:5’-ctcactataggTTGAGCCTCAACATCAATCG-3’;

km-galk-F:5’-TATTTCAGCActcaatcgaattacacagaaca-3’;

km-galk-R:5’-TTCTGGTTGccactagtggatctgatatcac-3’;

km-xks1-PUG6-F:5’-GAGGCTCAAcctatagtgagtcgtattaatttcg-3’;

km-galk-PUG6-R:5’-TTCTCTCGGTATATtatagtgtcacctaaatcgtatg-3’。

Kluyveromyces marxianus genome is used as a template, km-XKS1-upF and km-XKS1-upR, km-XKS1-doF and km-XKS1-doR are used as primers, and the upstream and downstream sequences of XKS1 are obtained through PCR amplification. The plasmid PUG6-XYL3-GALK is used as a template, km-galk-F, km-galk-R is used as a primer, and the sequence of the resistance sequence Galk-Kan is obtained through PCR amplification. The PUG6 plasmid skeleton is obtained by PCR amplification by taking the PUG6-XYL3-GALK plasmid as a template and km-xks1-PUG6-F, km-galk-PUG6-R as a primer. The plasmid in the positive transformant was obtained in the same manner as above and was designated as PUG6-km-XKS1-GALK2. The plasmid is used as a template, KM-XKS < 1 > -upF and KM-XKS < 1 > -doR are used as primers, and the KM-DeltaXKS1: GALK2-loxp-Kan-loxp expression cassette is obtained by amplification.

Construction of KM-DeltaPHA2: YQAB-loxp-kan-loxp expression cassette

Based on the sequence information of the PUG6-Xyl2-Yqab plasmid, the amplification primers were designed:

km-pha2-upF:5’-ggtgacactataATTCCATTTGGATTTGGCAG-3’;

km-pha2-upR:5’-gtaattcgattgagAGCTACTTTTAGTTGGAGATT-3’;

km-pha2-doF:5’-ccactagtggTTGTTGTTACTTACCATTCCG-3’;

km-pha2-doR:5’-cactataggCAATATGGCTACAGATATAGACC-3’;

km-yqab-kan-F:5’-AAAGTAGCTctcaatcgaattacacagaacac-3’;

km-yqab-kan-R:5’-GTAACAACAAccactagtggatctgatatcac-3’;

km-yqab-PUG6-F:5’-GTAGCCATATTGcctatagtgagtcgtattaatttcg-3’;

km-pha2-yqab-PUG6-R:5’-CCAAATGGAATtatagtgtcacctaaatcgtatg-3’。

Kluyveromyces marxianus genome is used as a template, km-PHA2-upF, km-PHA2-upR, km-PHA 2-doF) and km-PHA2-doR are used as primers, and the upstream and downstream sequences of PHA2 are obtained through PCR amplification. The plasmid PUG6-XYL2-YQAB containing Kan resistance is used as a template, km-yqab-Kan-F, km-yqab-Kan-R is used as a primer, and a resistance sequence Kan-Yqab common sequence is obtained through PCR amplification. The PUG6 plasmid skeleton is obtained by PCR amplification by taking the PUG6-XYL2-YQAB plasmid as a template and km-yqab-PUG6-F, km-pha2-yqab-PUG6-R as a primer. Then, agarose gel electrophoresis was performed, E.coli transformation was performed, and the plasmid was extracted, and the plasmid was designated as PUG6-km-PHA2-YQAB. The plasmid PUG6-KM-PHA2-YQAB is used as a template, and KM-PHA2-upF and KM-PHA 2-doR) are used as primers for PCR amplification to obtain a KM-delta PHA2: ecYQAB-loxp-kan-loxp expression cassette or a KM-delta PHA2: ddYQAB-loxp-kan-loxp expression cassette.

Construction of GALT mutant

The point mutation kit is utilized to respectively mutate the catalytic active sites of KM-GALT to construct three strains KL-GALT ^M1,KL-GALT^M2 and KL-GALT ^M3, and the amino acid residues of C54A/N166A/H179A/H294A/Q338A, C51A/H121A/195EA/H313A/E332A or H177A/H311A/K326A/F327A/V329A are mutated on the basis of SEQ ID NO: 6.

9. LNB synthetic strain construction and verification

The KM-DeltaGALT:: loxp-Kan-loxp knockout cassette and the KM-DeltaGALT:: LNBP1-loxp-Kan-loxp expression cassette are respectively transformed into Kluyveromyces marxianus and G418 resistance is deleted to obtain strains StrainKM-LNB-0 and StrainKM-LNB-1 respectively. The resistance of G418 in the KM-LNB-1 strain was deleted, and the KM-DeltaXYL 1:: LNBP1-loxp-Kan-loxp expression cassette was transformed into StrainKM-LNB-1 and G418 resistance was deleted to obtain strain StrainKM-LNB-2. The KM-. DELTA.XKS1: GALK-loxp-Kan-loxp expression cassette was transformed into StrainKM-LNB-2 and G418 resistance was deleted, yielding strain StrainKM-LNB-3. The KM-. DELTA.PHA2:. EcYQAB-loxp-kan-loxp expression cassette and the KM-. DELTA.PHA2:. DdYQAB-loxp-kan-loxp expression cassette were transformed into StrainKM-LNB-3 and G418 resistance was deleted, respectively, to give strains StrainKM-LNB-4 and StrainKM-LNB-5. The expression cassettes KM-DeltaXYL 1:: LNBP1-loxp-Kan-loxp, KM-DeltaXKS 1:: GALK-loxp-Kan-loxp and KM-DeltaPHA 2::: ecYQAB-loxp-Kan-loxp are sequentially introduced into Kluyveromyces marxianus, and are respectively introduced into KM-GALT ^M1,KM-GALT^M2,KM-GALT^M3 to obtain strains StrainKM-LNB-6, strainKM-LNB-7 and StrainKM-LNB-8. The strain StrainKM-LNB-9 is obtained by sequentially introducing KM-DeltaXYL 1:: LNBP1-loxp-Kan-loxp expression cassette and KM-DeltaPHA 2::: ecYQAB-loxp-Kan-loxp into Kluyveromyces marxianus and deleting G418 resistance. The strain StrainKM-LNB-10 is obtained by sequentially introducing KM-DeltaXYL 1:: LNBP1-loxp-Kan-loxp expression cassette and KM-DeltaPHA 2::: ddYQAB-loxp-Kan-loxp into Kluyveromyces marxianus and deleting G418 resistance.

The KM-DeltaGALT:: LNBP2-loxp-Kan-loxp expression cassette was transformed into Kluyveromyces marxianus and G418 resistance was deleted to obtain strain StrainKM-LNB-1-1. The KM-DeltaXYL 1:LNBP 2-loxp-Kan-loxp expression cassette was transformed into StrainKM-LNB-1-1 and G418 resistance was deleted to give strain StrainKM-LNB-2-1. The KM-. DELTA.XKS1: GALK-loxp-Kan-loxp expression cassette was transformed into StrainKM-LNB-2 and G418 resistance was deleted to give strain StrainKM-LNB-3-1.

KM-DeltaPHA2: ecYQAB-loxp-kan-loxp was introduced into Kluyveromyces marxianus StrainKM-LNB-3-1 and G418 resistance was deleted to obtain strain StrainKM-LNB-4-1. KM-DeltaPHA2: ddYQAB-loxp-kan-loxp was introduced into Kluyveromyces marxianus StrainKM-LNB-3-1 and G418 resistance was deleted to obtain strain StrainKM-LNB-5-1. Introducing the expression cassette of KM-DeltaXYL 1:LNBP 2-loxp-Kan-loxp and the expression cassette of KM-DeltaPHA 2: ecYQAB-loxp-Kan-loxp into a Kluyveromyces marxianus starting strain and deleting G418 resistance to obtain a strain StrainKM-LNB-6-1; the strain StrainKM-LNB-7-1 is obtained by introducing the LNBP2-loxp-Kan-loxp expression cassette and the KM- ΔPHA2: ddYQAB-loxp-Kan-loxp expression cassette into Kluyveromyces marxianus starting strain, the strain StrainKM-LNB-8-1 is obtained by introducing the KM- ΔXYL1:LNBP 2-loxp-Kan-loxp expression cassette, the KM- ΔXKS1: GALK-loxp-Kan-loxp expression cassette and the KM- ΔPHA2: ecYQAB-loxp-Kan-loxp expression cassette into Kluyveromyces marxianus starting strain, and the strain StrainKM-LNB-8-1, the strain StrainKM-LNB-9-1 and the strain StrainKM-LNB-10-1 are obtained by introducing the Kluyveromyces marxianus starting strain. The KM-DeltaPHA2: ecYQAB-loxp-kan-loxp expression cassette and KM-DeltaPHA2: ddYQAB-loxp-kan-loxp were introduced into Kluyveromyces marxianus StrainKM-LNB-0, respectively, and G418 resistance was deleted to obtain strains StrainKM-LNB-11 and StrainKM-LNB-12.

The genetic engineering strains and the genotypes of the strains are shown in Table 2. To verify the correctness of the above strains, we extracted the genomes of the above transformants and the original strain, and amplified the genomes by PCR using the corresponding knockout or expression cassette amplification primers. And if the size of the unique band obtained after PCR amplification is consistent with that of the knockout box or the expression box, judging that the strain is correct, otherwise, judging that the strain is false positive.

TABLE 2 Kluyveromyces marxianus recombinant strain and genotype thereof

EXAMPLE 3 Synthesis of LNB by fermentation of Kluyveromyces lactis genetically engineered bacterium and Kluyveromyces marxianus genetically engineered bacterium

Lactose or glucose and galactose are used as substrates, and a two-stage culture is used for carrying out LNB synthesis verification experiments by using Kluyveromyces lactis cells, kluyveromyces marxianus cells, the Kluyveromyces lactis genetically engineered bacteria obtained in example 1 and the Kluyveromyces marxianus genetically engineered bacteria obtained in example 2 respectively.

1. Fermentation experiment:

In the first stage, glucose is used as carbon source to culture yeast for fast growth, and the yeast growth enters the final logarithmic phase or the stationary phase. Culturing strains in solid culture medium such as YDP at 30deg.C for 2-3 days, picking single colony, inoculating to 1.5mLYPD liquid culture medium, and shake culturing at 30deg.C and 200 rpm overnight. Then, the cells were inoculated in 50mL liquid medium shake flasks at an inoculum size of 2%, cultured at 30℃and 200 rpm until OD ₆₀₀ =1, and inoculated in 5L YPD medium (yeast extract 10g/L, peptone 20g/L, glucose 20 g/L) (10L volume fermenter) at an inoculum size of 2% (30 ℃) and 200 rpm shaking culture to accumulate the cell size.

In the second stage, when the product is synthesized at 30 ℃ and cultured under shaking at 200 rpm ℃ to 40 h, 20g/L lactose is fed in or 10 g/L glucose and 10 g/L galactose are used for replacing 20g/L lactose, and the total fermentation time is 72 h. After fermentation 72 h is finished, centrifuging the fermentation liquor, respectively collecting supernatant and precipitate, crushing the precipitate by using a high-pressure homogenizing crusher, boiling and centrifuging to remove protein, and combining the supernatant to obtain a final yeast fermentation product.

2. Identification of the product

(1) Standards and fermentation broth were tested according to the HPLC analysis method described above. HPLC analysis showed that LNB standard had a peak time rt of 10.5 min.

The Kluyveromyces marxianus genetically engineered bacteria obtained in example 1 and the fermentation broth of the Kluyveromyces marxianus genetically engineered bacteria obtained in example 2 all have strong absorption peaks near 10.5 min, and the peak time is consistent with that of an LNB standard substance, so that the fermentation of the yeast genetically engineered bacteria is primarily demonstrated to generate lactose-N-disaccharide (LNB).

(2) LC-MS analysis

The product of rt=10.5 min in the HPLC profile was subjected to LC-MS analysis. The conditions for LC-MS analysis are AminexHPX-87H (Bio-Rad, inc., hercules.) and a detector, namely an ultraviolet detector (Hitachi Chromaster), the detection wavelength is 210nm, the sample injection amount is 10 mu L, the flow rate is 0.5ml/min, the column temperature is 45 ℃, the mobile phase is 5mM sulfuric acid aqueous solution, and the molecular weight scanning range is 400-900 in the H-ESI mode.

LC-MS spectra show that LNB standard molecular weight 384.1459 should be lnb+h and molecular weight 406.1315 should be lnb+na. The Mass spectrum results can be retrieved as follows: compound CID 440994,Chemcial Formula:C ₁₄H₂₅NO₁₁, extract Mass 383.1.

The LC-MS profile of the rt=10.5 min species in the HPLC profile of the fermentation product was consistent with that of the LNB standard. The material in the broth with rt=10.5 min is illustrated as LNB.

In summary, the molecular weight of the product obtained by fermenting the yeast genetically engineered bacterium of the invention is about 383, and the peak time is consistent with the peak time of the LNB standard, namely, the fermentation solution obtained by the invention contains lactose-N-disaccharide (LNB, molecular weight 383.1).

The LNB yield in the fermentation broth was measured by HPLC analysis as described above and the results are reported in tables 3 and 4, respectively.

As determined by analysis:

(1) The kluyveromyces lactis starting strain ,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、StrainKL-LNB-12 is a genetically engineered strain which does not produce LNB.

(2) The kluyveromyces marxianus starting strain ,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、StrainKM-LNB-12 genetically engineered strain did not produce LNB.

(3) The results of LNB production by other genetically engineered strains are reported in tables 3 and 4, respectively.

TABLE 3 research on LNB production by Kluyveromyces lactis genetically engineered bacteria

TABLE 4 LNB research of Kluyveromyces marxianus genetically engineered bacteria

。

The data in tables 3-4 illustrate that in the Kluyveromyces lactis or Kluyveromyces marxianus strain:

(1) After lactose-N-disaccharide phosphorylase (LNBP) and fructose-1-phosphate phosphorylase (YQAB) are introduced simultaneously, LNB can be synthesized, and even if milk-N-disaccharide phosphorylase (LNBP) with different sources and fructose-1-phosphate phosphorylase (YQAB) with different sources are introduced, LNB can still be synthesized;

(2) On the basis of (1), the yield of LNB is greatly improved by destroying a marker GALT gene of yeast or modifying an amino acid sequence of GalT.

(3) The resulting genetically engineered strain has no capacity to synthesize LNB by introducing lactose-N-disaccharide phosphorylase (LNBP) or fructose-1-phosphate phosphorylase (YQAB) alone or disrupting the yeast marker GALT gene or modifying the yeast marker GALT gene.

(4) The LNB yields were not greatly different whether lactose or a mixed sugar of galactose and glucose was used as the carbon source.

Example 4 construction of molecular switch-regulated LNB production in Kluyveromyces lactis

Construction of KL-Delta GALE:P _tef -CAMR-Tuap-PcamO-AGM1-Tagm-loxp-Kan-loxp expression cassette:

And (3) taking the kluyveromyces lactis genome as a template, and performing PCR amplification to obtain upstream and downstream sequences of GalE. The pUG6 plasmid containing Kan resistance is used as a template, and the resistance sequence Kan is obtained through PCR amplification. And (3) taking the PUG6 plasmid as a template, and carrying out PCR amplification to obtain a PUG6 plasmid skeleton. Agarose gel electrophoresis is carried out after PCR amplification is finished, the target band is recovered and then connected by using 2 x MultiF Seamless Assembly Mix, all fragments are added according to equimolar number, and after connection for 30min at 50 ℃, escherichia coli transformation is carried out. The positive E.coli transformants were picked for plasmid extraction and the plasmid was designated PUG6-GALE. The PUG6-GALE plasmid is used as a template, and the PUG6-GalE plasmid skeleton is obtained through PCR amplification. Then amplifying to obtain a promoter tef by taking a kluyveromyces lactis genome as a template, amplifying by taking a genetically synthesized transcription factor CAMR as the template, obtaining a transcription factor CAMR sequence by PCR amplification, wherein the transcription factor CAMR sequence is shown as SEQ ID NO.7, obtaining a UAP termination sequence by taking the kluyveromyces lactis genome as the template, obtaining a promoter CamO by PCR amplification by taking a genetically synthesized CamR response type promoter sequence as the template, and obtaining a CDS sequence and a termination sequence of AGM1 by taking the kluyveromyces lactis genome as the template, wherein the CDS sequence and the termination sequence of AGM1 are shown as SEQ ID NO. 9. The plasmid skeleton of PUG6-GALE, the promoter tef, the transcription factor CAMR, the termination sequence of UAP, the promoter CamO sequence, the CDS sequence of AGM1 and the termination sequence band thereof are recovered and then connected for Escherichia coli transformation. Positive transformants were picked for plasmid extraction and the plasmid was designated PUG 6-. DELTA. GALE-CAMR-AGM1. The plasmid PUG 6-Delta GALE-CAMR-AGM1 is used as a template, galE up-s and GalE down-a are used as primers for PCR amplification to obtain the KL-Delta GALE, namely the P _tef -CAMR-Tuap-PcamO-AGM1-Tagm-loxp-Kan-loxp expression cassette. The primers were designed as follows:

GalE up-s: 5’-ctatagaacgcATGTCTGAAGATAAATACTGTTTGGT-3’;

GalE up-a: 5’-gttgtcgacctgcaCAGATGGATGAGCACCAATAG-3’。

Construction of KL-delta AGM 1:loxp-Kan-loxp knockout cassette

According to the upstream and downstream sequences of AGM1, the upstream and downstream sequences of AGM1 are obtained by PCR amplification by taking the Kluyveromyces lactis genome as a template. The pUG6 plasmid containing Kan resistance is used as a template, and the resistance sequence Kan is obtained through PCR amplification. And (3) PCR amplification to obtain the PUG6 plasmid skeleton. The target band was recovered by agarose gel electrophoresis, E.coli transformation was performed after ligation, and the plasmid was extracted and designated as PUG6-AGM1. And (3) carrying out PCR amplification by taking the PUG6-AGM1 plasmid as a template and the AGM1 up-s and AGM1down-a as primers to obtain the KL-delta AGM1 i.e. loxp-Kan-loxp knockout box. The primers were designed as follows:

AGM1 up-s:5’-cactatagaacgcgttgcgaagtctcatctgtt-3’;

AGM1 up-a:5’-tgtcgacctgcagtcacggaaagacattggtg-3’。

3. construction and verification of strain for regulating LNB (Low-Density B) generation by molecular switch

Construction of StrainKL-LNB-13 Strain KL-DeltaAGM 1: loxp-Kan-loxp knockout cassette and KL-DeltaGalE:: P _tef -CAMR-Tuap-PcamO-AGM1-Tagm-loxp-Kan-loxp expression cassette were transformed into Kluyveromyces lactis StrainKL-LNB-4 strain, and after G418 resistance was deleted, strainKL-LNB-13 strain was obtained.

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 or expression cassette amplification primers. And if the size of the unique band obtained after PCR amplification is consistent with that of the knockout box or the expression box, judging that the strain is correct, otherwise, judging that the strain is false positive.

Example 5 construction of molecular switch-regulated LNB production in Kluyveromyces marxianus

Construction of the KM-Delta GALE:P _tef -CAMR-Tuap-PcamO-AGM1-Tagm-loxp-Kan-loxp expression cassette

The kluyveromyces marxianus genome is used as a template, and the upstream and downstream sequences of GALE are obtained through PCR amplification. The pUG6 plasmid containing Kan resistance is used as a template, and the resistance sequence Kan is obtained through PCR amplification. And (3) taking the PUG6 plasmid as a template, and carrying out PCR amplification to obtain a PUG6 plasmid skeleton. And (3) recovering the target band through agarose gel electrophoresis, and then connecting and converting. The positive E.coli transformants were picked for plasmid extraction and the plasmid was designated PUG6-GALE. And (3) taking the PUG6-GalE plasmid as a template, and carrying out PCR amplification to obtain the PUG6-GALE plasmid skeleton. Then, using Kluyveromyces marxianus genome as a template, amplifying to obtain a promoter tef, using a genetically synthesized transcription factor CAMR as a template, performing PCR amplification to obtain a transcription factor CAMR sequence, using the Kluyveromyces marxianus genome as the template, performing PCR amplification to obtain a UAP termination sequence, using a genetically synthesized CAMR response type promoter sequence as the template, performing PCR amplification to obtain a promoter CamO, using the Kluyveromyces marxianus genome as the template, and performing PCR amplification to obtain a CDS sequence of AGM1 and a termination sequence. The PUG6-GALE plasmid backbone, promoter tef, transcription factor CAMR, termination sequence of UAP, promoter CamO sequence, CDS sequence of AGM1 and its termination sequence band were recovered and then ligated using 2 x MultiF Seamless Assembly Mix, transformed into e. Positive transformants were picked for plasmid extraction and the plasmid was designated PUG 6-. DELTA. GALE-CAMR-AGM1. The KM-delta GALE:. Sub. _tef -CAMR-Tuap-PcamO-AGM1-Tagm-loxp-Kan-loxp expression cassette is obtained by PCR amplification with the plasmid PUG 6-delta GALE-CAMR-AGM1 as template, galE up-s and GalE down-a as primers. The primers were designed as follows:

GalE up-s:5’-ctatagaacgcATGTCTGAAGATAAATACTGTTTGGT-3’;

GalE up-a:5’-gttgtcgacctgcaCAGATGGATGAGCACCAATAG-3’。

construction of a KM-DeltaAGM 1:loxp-Kan-loxp knockout cassette:

according to the upstream and downstream sequences of AGM1, the upstream and downstream sequences of AGM1 are obtained by PCR amplification using Kluyveromyces marxianus genome as a template. The pUG6 plasmid containing Kan resistance is used as a template, and the resistance sequence Kan is obtained through PCR amplification. And (3) PCR amplification to obtain the PUG6 plasmid skeleton. The fragments were subjected to agarose gel electrophoresis, and after the target band was recovered, the fragments were ligated to carry out E.coli transformation. Positive transformants were picked for plasmid extraction and the plasmid was designated as PUG6-AGM1. And (3) carrying out PCR amplification by taking the PUG6-AGM1 plasmid as a template and AGM1 up-s and AGM1down-a as primers to obtain the KM-delta AGM1 i.e. loxp-Kan-loxp knockout box. The primers were designed as follows:

AGM1 up-s:5’-cactatagaacgcgttgcgaagtctcatctgtt-3’;

AGM1 up-a:5’-tgtcgacctgcagtcacggaaagacattggtg-3’。

Construction of StrainKM-LNB-13 Strain KM-DeltaAGM 1: loxp-Kan-loxp knockout cassette and KM-Delta GALE:: P _tef -CAMR-Tuap-PcamO-AGM1-Tagm-loxp-Kan-loxp expression cassette were transformed into Kluyveromyces marxianus LNB-4 strain, and StrainKM-LNB-13 strain was obtained after G418 resistance was deleted.

EXAMPLE 6 fermentation experiments of Kluyveromyces marxianus genetically engineered bacteria obtained in examples 4-5

The synthesis verification experiment of LNB is carried out by adopting 20g/L lactose or 10 g/L glucose and 10 g/L galactose as carbon sources, camphor as a molecular switch regulating small molecule and adopting two-stage culture.

1. Fermentation experiment

The first stage is the bacterial growth period, and the first stage of the fermentation experiment is the same as that of the embodiment 3.

The second stage is a product synthesis stage, in which 20 g/L lactose or 10 g/L glucose and 10 g/L galactose are fed in when shaking culture is performed at 30 ℃ under 200 rpm until 40 h, and 200 mu M camphor is added for fermentation for 72 hours. After fermentation for 72 hours, centrifuging the fermentation liquor, respectively collecting supernatant and sediment, crushing the sediment by using a high-pressure homogenizing crusher, boiling and centrifuging to remove protein, and combining the supernatant to obtain a final yeast fermentation product.

2. The identification and content analysis of the products were carried out in the same manner as in example 3, and the LNB production amounts of the yeast genetically engineered strains were recorded separately from those of Table 5.

TABLE 5 Synthesis of LNB by Kluyveromyces lactis and Kluyveromyces marxianus genetically engineered strain

。

The data in Table 5 shows that the metabolic flux in yeast is changed by adding small molecular substances with the molecular switch regulating and controlling function in the stationary phase after the growth of the Kluyveromyces lactis genetic engineering strain and the Kluyveromyces marxianus genetic engineering strain before the introduction of the molecular switch, so that the LNB yield is greatly improved by nearly ten times.

Although the invention has been described in considerable detail by way of illustration and example for the purpose of clarity of understanding, it will be apparent to those of ordinary skill in the art that any equivalent aspects or modifications may be practiced. Accordingly, the specification and examples should not be construed as limiting the scope of the invention.

Claims

1. A yeast genetic engineering bacterium is characterized by comprising genes of heterologous lactose-N-disaccharide phosphorylase and fructose-1-phosphate phosphorylase, wherein the yeast genetic engineering bacterium is selected from Kluyveromyces lactis and Kluyveromyces marxianus, and can synthesize lactose-N-disaccharide by taking a culture medium containing lactose or galactose as a raw material,

The lactose-N-disaccharide phosphorylase gene is derived from bifidobacterium longum Bifidobacteirum longum, the amino acid sequence of which is shown as SEQ ID NO. 1, or from Bacillus cereus, the amino acid sequence of which is shown as SEQ ID NO. 2,

The fructose-1-phosphate phosphorylase gene is derived from Escherichia coli ESCHERICHIA COLI, the amino acid sequence of which is shown in SEQ ID NO. 3 or from bacterial sweet potato stem rot germ DICKEYA DADANTII, and the amino acid sequence of which is shown in SEQ ID NO. 4.

2. The yeast genetic engineering bacterium of claim 1, further comprising disruption of a yeast marker gene, wherein the amino acid sequence of the marker gene of kluyveromyces lactis is shown in SEQ ID No. 5, and the amino acid sequence of the marker gene of kluyveromyces marxianus is shown in SEQ ID No. 6.