Recombinant microorganisms and their use in the production of 2'-fucosyllactose

Abstract

The application belongs to the technical field of biology, and provides a recombinant microorganism and application of the recombinant microorganism in preparation of 2'-fucosyllactose, wherein the recombinant microorganism carries nucleic acids encoding GDP-mannose dehydratase, GDP-L-fucose synthase and alpha-(1,2) fucosyltransferase. It is found that by making the microorganism express GDP-mannose dehydratase, GDP-L-fucose synthase and alpha-(1,2) fucosyltransferase and adjusting the copy number of the above genes, efficient production of 2'-fucosyllactose can be realized, and the production cost is low, the cycle is short, and the yield is high.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biotechnology, and in particular, the present application relates to a recombinant microorganism and its application in preparing 2'-fucosyllactose. BACKGROUND

[0002] Human milk oligosaccharides are unique nutritional components in human milk, and are the third largest nutrient after lactose and fat. 2'-fucosyllactose (2'-FL) is the main functional component of human milk oligosaccharides, and plays an important role in infant growth and development, immune system development, intestinal microecosystem establishment, and intestinal mucosal barrier function. The human milk oligosaccharides isolated from human milk currently have certain applications in structural identification and basic research, but due to their extremely low yield, the research on their biological effects and application in infant nutrition are hindered. Therefore, it is particularly important to explore and develop artificial synthesis of human milk oligosaccharides.

[0003] Currently, the direct extraction and chemical synthesis of 2'-fucosyllactose have low yield and many limitations. Biological synthesis is the main method for industrial production. Through genetic engineering means, the metabolic pathways of the biological chassis are modified to express various reaction enzymes and produce 2'-fucosyllactose. For example, the yield of 2'-fucosyllactose synthesized by using Escherichia coli as a chassis is high, but there are many problems in biological safety due to the presence of endotoxins and the easy contamination of phages. Another industrial microorganism, Saccharomyces cerevisiae, has been widely used in genetic engineering research such as gene knockout, gene silencing, and gene overexpression. For a long time, Saccharomyces cerevisiae has been used as a general industrial microorganism strain to synthesize various biological elements and biomolecules. So far, more than 100 natural products have been synthesized using Saccharomyces cerevisiae as a chassis. As a eukaryotic chassis host, Saccharomyces cerevisiae has a similar structure to plant cells, such as the endoplasmic reticulum system that microsomal enzymes rely on to survive, and has become a widely used host for producing important compounds.

[0004] Traditional metabolic engineering usually involves overexpression of genes encoding rate-limiting enzymes, reduction of expression of non-essential genes, knockout of competing pathways, and introduction of new biosynthetic pathways, but lacks regulation of the content of different enzymes. In addition, traditional metabolic engineering of Saccharomyces cerevisiae requires the use of homologous recombination to introduce foreign fragments into the genome, which has the disadvantages of low efficiency, large workload, and low throughput, which makes the process of constructing recombinant strains long and increases the research and development cost. Therefore, there is still a need to develop a low-cost, short-cycle, and high-yield method for producing 2'-fucosyllactose. SUMMARY

[0005] The present application aims to at least partially solve one of the technical problems in the related art.

[0006] GDP-mannose dehydratase, GDP-L-fucose synthase and α-(1,2) fucosyltransferase constitute the core enzymatic machinery of 2'-FL biosynthesis. GDP-mannose dehydratase (GMD) is usually the most important rate-limiting enzyme in the whole pathway, limiting the supply of GDP-fucose precursor. The efficiency of GDP-L-fucose synthase (GMER) affects the conversion rate and stability of intermediates, and also restricts the effective output of GDP-fucose. The efficiency of α-(1,2) fucosyltransferase (FutC) directly determines whether GDP-fucose can be efficiently and specifically converted into target product 2'-FL. Optimizing the activity, expression and stability of the three enzymes through metabolic engineering methods (such as gene overexpression, cofactor engineering, competitive pathway knockout) and protein engineering (enzyme modification) is the core strategy to improve the yield of 2'-FL in microbial cell factories. Identifying which (or which) is the main bottleneck in a particular engineered strain is the key to further optimization. Saccharomyces cerevisiae is a commonly used chassis strain for biosynthesis, but there is currently no work on quantitatively adjusting the expression of the three enzymes in Saccharomyces cerevisiae to regulate the final product 2'-FL.

[0007] Therefore, the present application optimizes and modifies the recombinant microorganism producing 2'-fucosyllactose, and significantly improves the production efficiency of 2'-fucosyllactose, shortens the production cycle, and reduces the production cost, by overexpressing the coding nucleic acids and copy numbers of GDP-mannose dehydratase, GDP-L-fucose synthase and α-(1,2) fucosyltransferase. The yield of 2'-fucosyllactose can be significantly improved by optimizing the copy number and culture method.

[0008] Therefore, according to the first aspect of the present application, the present application provides a recombinant microorganism. According to embodiments of the present application, the recombinant microorganism carries nucleic acids encoding GDP-mannose dehydratase, GDP-L-fucose synthase and α-(1,2) fucosyltransferase. The expression levels of GDP-mannose dehydratase, GDP-L-fucose synthase and α-(1,2) fucosyltransferase of the recombinant microorganism are up-regulated. In addition, the present application adjusts the arrangement order of each gene on the plasmid, the strength of the promoter and other means to make the expression amounts of GDP-mannose dehydratase, GDP-L-fucose synthase and α-(1,2) fucosyltransferase reach a relatively balanced and efficient synergistic working ratio, thereby significantly improving the production efficiency of 2'-fucosyllactose of the recombinant microorganism, shortening the production cycle and reducing the production cost.

[0009] According to embodiments of the present application, the above-mentioned recombinant microorganism comprises at least one of the following additional technical features:

[0010] According to an embodiment of the present application, the copy number of the nucleic acid encoding GDP-mannose dehydratase is 2-3, the copy number of the nucleic acid encoding GDP-L-fucose synthase is 2-3, and the copy number of the nucleic acid encoding alpha-(1,2) fucosyltransferase is 2-3. The present application controls the yield and production efficiency of 2'-fucosyllactose by adjusting the copy number of the above-mentioned genes in the genome of the recombinant microorganism, and the microorganism can efficiently produce 2'-fucosyllactose when the copy number is within the range, with low production cost and short production cycle.

[0011] According to a preferred embodiment of the present application, the copy number of the nucleic acid encoding GDP-mannose dehydratase is 3, the copy number of the nucleic acid encoding GDP-L-fucose synthase is 3, and the copy number of the nucleic acid encoding alpha-(1,2) fucosyltransferase is 2.

[0012] According to an embodiment of the present application, the expression level of lactose transporter of the recombinant microorganism is up-regulated to achieve the transport of lactose in the culture medium to the intracellular for 2'FL synthesis; and the expression level of oligosaccharide transport protein of the recombinant microorganism is up-regulated to achieve the transport of the synthesized 2'FL to the extracellular function.

[0013] According to an embodiment of the present application, the recombinant microorganism carries nucleic acid encoding lactose transporter and / or oligosaccharide transport protein.

[0014] According to an embodiment of the present application, the copy number of the nucleic acid encoding lactose transporter is 1-3 and / or the copy number of the nucleic acid encoding oligosaccharide transport protein is 1-3.

[0015] According to an embodiment of the present application, the nucleic acid encoding GDP-mannose dehydratase, GDP-L-fucose synthase and alpha-(1,2) fucosyltransferase is inserted at the chromosome Chr XII-4 site. It should be noted that the chromosome ChrXII-4 site is located by gRNA shown in SEQ ID NO: 65.

[0016] According to an embodiment of the present application, the nucleic acid encoding lactose transporter is inserted at the chromosome ChrXI-3 site, and / or the nucleic acid encoding oligosaccharide transport protein is inserted at the chromosome ChrX-3 site.

[0017] According to an embodiment of the present application, the nucleic acid encoding the alpha-(1,2) fucosyltransferase can also be inserted at the chromosome ChrXIV-Int10 and / or ChrXVI-Int14 site.

[0018] According to an embodiment of the present application, the nucleic acid encoding the GDP-mannose dehydratase and / or the GDP-L-fucose synthase can also be inserted into the ChrX-2 and / or Chrll-Intl site.

[0019] According to an embodiment of the present application, the nucleic acid sequences encoding the GDP-mannose dehydratase, the GDP-L-fucose synthase, the a-(l,2) fucosyltransferase, the lactose transporter and the oligosaccharide transporter protein are derived from at least one of Escherichia coli, Helicobacter pylori, Kluyveromyces lactis and Neurospora crassa.

[0020] According to an embodiment of the present application, the nucleic acid sequence encoding the GDP-mannose dehydratase and the nucleic acid sequence encoding the GDP-L-fucose synthase are derived from Escherichia coli, and / or the nucleic acid sequence encoding the a-(l,2) fucosyltransferase is derived from Helicobacter pylori, and / or the nucleic acid sequence encoding the lactose transporter is derived from Kluyveromyces lactis, and / or the nucleic acid sequence encoding the oligosaccharide transporter protein is derived from Neurospora crassa. It is understood by those skilled in the art that different microbial strains of different origins have different metabolic pathways and growth characteristics. When the proteins or their encoding nucleic acids in the microorganism are derived from different microorganisms, they have different compatibility with the expression system of the host strain; secondly, the nucleic acids of different origins can have differences in sequence composition. In the process of gene cloning and expression, if there are some sequences in the nucleic acid sequence that are not conducive to replication or transcription in the host microorganism, it will affect the yield of the nucleic acid product. When the encoding nucleic acid sequences of the respective proteins are of the above-mentioned origins, the yield and production efficiency of 2'-fucosyllactose are significantly improved.

[0021] According to an embodiment of the present application, the exogenous nucleic acid is operably linked to a promoter and / or a terminator, the promoter comprising at least one selected from the group consisting of Saccharomyces cerevisiae promoters pHXT7, pPDC1, pTEF2, pADH1 and pPYK1, and the terminator comprising at least one selected from the group consisting of Saccharomyces cerevisiae terminators tPGK1, tTEF1, tFBA1, tCYC1 and tADH1. The naming of the promoter and the terminator is the conventional naming manner in the art, and the selection of the used promoter and terminator directly affects the expression efficiency and quality of the target protein. The above-mentioned promoter and terminator are obtained after screening and optimization, the sequence characteristics of the transcription product of the promoter should match the coding sequence of the target gene to ensure efficient translation, and the promoter sequence remains stable during the growth and culture of the microorganism and does not mutate or lose. The promoter and the terminator are both adapted to the microbial expression system of the present application.

[0022] According to an embodiment of the present application, the genome of the recombinant microorganism has integrated the following nucleic acid fragments in series in tandem:

[0023] (1) pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1-pTEF2-FutC-tFBA1,

[0024] (2) pADH1-Lac12-tCYC1, and

[0025] (3) pPYK1-CDT2-tADH1.

[0026] According to an embodiment of the present application, the genome of the recombinant microorganism further integrates any one of the following in series:

[0027] (4) pTEF2-FutC-tFBA1,

[0028] (5) pTEF2-FutC-tFBA1 and pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1,

[0029] (6) pTEF2-FutC-tFBA1, pTEF2-FutC-tFBA1 and pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1,

[0030] (7) pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1, pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1 and pTEF2-FutC-tFBA1, and

[0031] (8) pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1, pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1, pTEF2-FutC-tFBA1 and pTEF2-FutC-tFBA1.

[0032] According to an embodiment of the present application, the genome of the recombinant microorganism further integrates any one of the following in series:

[0033] (A) inserting pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1-pTEF2-FutC-tFBA1 at the Chr XII-4 site, inserting pADH1-Lac12-tCYC1 at the Chr XI-3 site, and inserting pPYK1-CDT2-tADH1 at the Chr X-3 site,

[0034] (B) insertion of pHXT7-GMER-tPGKl-pPDC1-GMD-tTEFl-pTEF2-FutC-tFBA1 at the Chr XII-4 locus, insertion of pADHl-Lacl2-tCYCl at the Chr XI-3 locus, insertion of pPYKl-CDT2-tADHl at the Chr X-3 locus, insertion of pTEF2-FutC-tFBA1 at the Chr XIV-IntlO locus, insertion of pHXT7-GMER-tPGKl-pPDC1-GMD-tTEFl at the Chr X-2 locus,

[0035] (C) insertion of pHXT7-GMER-tPGKl-pPDC1-GMD-tTEFl-pTEF2-FutC-tFBA1 at the Chr XII-4 locus, insertion of pADHl-Lacl2-tCYCl at the Chr XI-3 locus, insertion of pPYKl-CDT2-tADHl at the Chr X-3 locus, insertion of pTEF2-FutC-tFBA1 at the Chr XIV-IntlO locus, insertion of pHXT7-GMER-tPGKl-pPDC1-GMD-tTEFl at the Chr X-2 locus,

[0036] (D) insertion of pHXT7-GMER-tPGKl-pPDC1-GMD-tTEFl-pTEF2-FutC-tFBA1 at the Chr XII-4 locus, insertion of pADHl-Lacl2-tCYCl at the Chr XI-3 locus, insertion of pPYKl-CDT2-tADHl at the Chr X-3 locus, insertion of pTEF2-FutC-tFBA1 at the Chr XIV-IntlO locus, insertion of pHXT7-GMER-tPGKl-pPDC1-GMD-tTEFl at the Chr X-2 locus, insertion of pTEF2-FutC-tFBA1 at the Chr XVI-Intl4 locus,

[0037] (E) inserting pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1-pTEF2-FutC-tFBA1 at the site of chromosome Chr XII-4, inserting pADH1-Lac12-tCYC1 at the site of chromosome Chr XI-3, inserting pPYK1-CDT2-tADH1 at the site of chromosome Chr X-3, inserting pTEF2-FutC-tFBA1 at the site of chromosome Chr XIV-Int10, inserting pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1 at the site of chromosome Chr X-2, inserting pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1 at the site of chromosome Chr II-Int1, and

[0038] (E) inserting pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1-pTEF2-FutC-tFBA1 at the site of chromosome Chr XII-4, inserting pADH1-Lac12-tCYC1 at the site of chromosome Chr XI-3, inserting pPYK1-CDT2-tADH1 at the site of chromosome Chr X-3, inserting pTEF2-FutC-tFBA1 at the site of chromosome Chr XIV-Int10, inserting pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1 at the site of chromosome Chr X-2, inserting pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1 at the site of chromosome Chr II-Int1, and inserting pTEF2-FutC-tFBA1 at the site of chromosome Chr XVI-Int14.

[0039] According to an embodiment of the present application, the recombinant microorganism is Saccharomyces cerevisiae, Escherichia coli or Pichia pastoris.

[0040] In a second aspect of the present application, the present application provides use of the recombinant microorganism of the first aspect in the preparation of 2'-fucosyllactose.

[0041] In a third aspect of the present application, the present application provides a method for preparing 2'-fucosyllactose, characterized in that, comprising:

[0042] culturing the recombinant microorganism of the first aspect inoculated into a liquid medium; and

[0043] separating 2'-fucosyllactose from the culture product of the liquid culture.

[0044] According to an embodiment of the present application, the above-mentioned method for preparing 2'-fucosyllactose can further comprise at least one of the following additional technical features:

[0045] According to the embodiment of the present application, the inoculation amount of the recombinant microorganism in the inoculation process is 0.5%-5%.

[0046] According to the embodiment of the present application, the culture includes: when the recombinant microorganism is cultured to an OD600 value of 1-3, lactose is added, and the final concentration of lactose in the liquid culture medium is 0.40%-0.50%.

[0047] According to the embodiment of the present application, after the addition of lactose, the recombinant microorganism is further cultured for 20-28 hours to obtain 2'-fucosyllactose.

[0048] According to the embodiment of the present application, the culture is carried out at 28-32°C.

[0049] According to the embodiment of the present application, the liquid culture medium includes a basic culture medium and glucose, and the final concentration of glucose is 2%-4%.

[0050] According to the embodiment of the present application, the basic culture medium is an SD culture medium.

[0051] Compared with the prior art, the present application has at least the following beneficial technical effects:

[0052] (1) The present application optimizes the copy number of GDP-mannose dehydratase, GDP-L-fucose synthase and alpha-(1,2) fucosyltransferase, and unexpectedly finds that the production efficiency of the optimized recombinant microorganism 2'-fucosyllactose is significantly improved, and three key genes are introduced at one time in the construction process, which is simple and convenient to operate, low in cost and short in cycle;

[0053] (2) When the recombinant microorganism is used for fermentation culture in cooperation with the shake flask fermentation method developed in the present application, 2'-fucosyllactose can be produced in large quantities, and the yield is as high as 724.4 mg / L.

[0054] Additional aspects and advantages of the present application will be partially given in the following description, partially will become obvious from the following description, or will be understood by the practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0055] The above and / or additional aspects and advantages of the present application will become apparent and easy to understand from the description of the embodiments, combined with the following drawings, in which:

[0056] Figure 1 The figure shows the map of the 01 plasmid in Example 1 of the present application;

[0057] Figure 2 The figure shows the map of the 02 plasmid in Example 1 of the present application;

[0058] Figure 3 Figure showing the map of 03 plasmid in Example 1 of the present application;

[0059] Figure 4 Figure showing the map of 04 plasmid in Example 1 of the present application;

[0060] Figure 5 Figure showing the results of the effect of different copy numbers of each gene on 2-FL production in Example 2 of the present application. DETAILED DESCRIPTION

[0061] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar components are denoted by the same or similar reference numerals throughout. The embodiments described below by reference to the drawings are exemplary and are intended to explain the present application, and are not to be understood as limiting the present application.

[0062] DEFINITIONS

[0063] As used herein, the terms or expressions used herein have the following meanings unless otherwise specified.

[0064] The term "recombinant microorganism" as used herein refers to a microorganism which has been genetically modified or engineered so that the sequence or expression of an endogenous nucleic acid is altered, or an exogenous nucleic acid is expressed (the exogenous nucleic acid can be contained in a vector or integrated into the genome of the microorganism). By "altered" is meant that the expression of a gene, or the level of an RNA molecule, or the level of an equivalent RNA molecule encoding one or more polypeptides or polypeptide subunits, or the activity of one or more polypeptides or polypeptide subunits, is up- or down-regulated, such that the expression, level or activity is greater or less than that observed in the absence of the alteration.

[0065] The term "exogenous" as used herein refers to a substance that does not originally exist in the starting strain, or a substance that is distinguished from the corresponding substance in the starting strain, such as a nucleic acid that does not exist in the starting strain, or a nucleic acid that has a mutation compared to the corresponding gene sequence in the starting strain. According to the embodiments of the present application, "exogenous nucleic acid" includes nucleic acid from the same species as the starting strain, and also includes heterologous nucleic acid (i.e., nucleic acid from a different species than the starting strain).

[0066] The term "nucleic acid" as used herein is used interchangeably with the term "polynucleotide" and refers to an organic polymer composed of two or more monomers, including nucleotides, nucleosides or analogs thereof, and includes but is not limited to single- or double-stranded, sense or antisense deoxyribonucleic acid (DNA), sense or antisense ribonucleic acid (RNA) of any length, including siRNA. The term "nucleotide" refers to any one of a variety of compounds composed of ribose or deoxyribose combined with a purine or pyrimidine base and a phosphate group, and they are the basic structural units of nucleic acids. The term "nucleoside" refers to a compound composed of a purine or pyrimidine base combined with deoxyribose or ribose, such as guanosine or adenosine, and it exists in nucleic acids.

[0067] It should be understood that the meaning of "nucleic acid" described herein includes "gene", and the nucleic acid described herein also includes "vector" or "plasmid".

[0068] The term "gene" as used herein refers to a polynucleotide that encodes a specific amino acid sequence, which comprises all or part of one or more proteins or enzymes, and can include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine, for example, under what conditions the gene is expressed. The transcribed region of a gene can include untranslated regions (including introns, 5'-untranslated region (UTR) and 3'UTR) as well as coding sequences.

[0069] The term "up-regulation" as used herein refers to the activity of a specific functional protein being increased compared to the corresponding activity of the starting strain, which can be achieved by increasing the expression amount of the specific functional protein in the microbial cell (for example, additional introduction of an exogenous nucleic acid encoding the functional protein, which can also be achieved by adjusting the regulatory elements such as promoters of the functional protein encoding nucleic acid), or by improving the biological activity of the functional protein by modifying its amino acid sequence.

[0070] The term "expression" as used herein refers to the transcription and / or translation of a coding gene segment or gene. Typically, the resulting product is mRNA or protein. However, expression products can also include functional RNA, such as antisense strand nucleic acids, tRNA, snRNA, rRNA, RNAi, siRNA, ribozyme, etc. Expression can be global, local or transient, for example, limited to a specific cell type, tissue, organ or time phase.

[0071] The term "inserting into the corresponding site" as used herein refers to the additional introduction of other exogenous nucleic acids at the site of the original gene, so that the exogenous nucleic acid can be integrated into the genome of the microorganism to function.

[0072] The term "operably linked to a promoter" as used herein refers to a nucleic acid molecule linked to a promoter that can be controlled by the promoter.

[0073] The term "about" as used herein means a range of 5% above and below the index value. For example, a value of about 100 means the value can be 100 plus or minus 5.

[0074] In addition, the skilled person in the art can understand that after determining the gene site and the corresponding sequence that need to be modified, the skilled person in the art can use common molecular biology means to realize the modification or the modification of the specific gene. For example, the gene editing means can be used. The term "gene editing" as used herein refers to the modification (including but not limited to insertion, deletion or replacement) of a specific target gene (also referred to as "target gene" or "target") in the genome of an organism, according to the embodiments of the present application, for example, using CRISPR / Cas9 technology. Specifically, under the guidance of sgRNA (recognizing the target gene), the nuclease Cas9 as a "molecular scissors" is used to generate a site-specific double-strand break (DSB) at a specific position in the genome, and further induce the organism to repair the DSB through non-homologous end joining (NHEJ) or homologous recombination (HR) (also referred to as "HDR" (Homology directed repair, homology directed double-strand DNA repair)), thereby realizing the specific modification, wherein in order to improve the efficiency of homologous recombination mediated double-strand DNA repair (HDR), an additional "donor" or repair template based on the sequence of the target gene is added as a homologous recombination repair template, thereby realizing the purpose of replacing the sequence of the original genome at the specific position with the sequence of the donor.

[0075] It should be noted that the nucleic acid encoding GDP-mannose dehydratase (GMD) in the present application is derived from E. coli K-12 Escherichia coli K12, the nucleic acid encoding GDP-L-fucose synthase (GMER) is derived from Escherichia coli K12, the nucleic acid encoding α-(1,2) fucosyltransferase (FutC) is derived from Helicobacter pylori, the nucleic acid encoding lactose transporter (Lac12) is derived from Kluyveromyces lactis, and the nucleic acid encoding oligosaccharide transporter protein (CDT-2) is derived from Neurospora crassa.

[0076] In the present application, "in series" means direct and / or indirect connection according to the arrangement order.

[0077] In the present application, "2'-fucosyllactose", "2'-FUCOSYLLACTOSE", "2'-FL" and "2-FL" are synonymous, with a CAS number of 41263-94-9, a chemical formula of C 18 H 32 O 15 , the structure of which is as shown below:

[0078] .

[0079] It should be noted herein that the chromosome ChrXII-4 site is located by a gRNA shown in SEQ ID NO: 65, the chromosome ChrXI-3 site is located by a gRNA shown in SEQ ID NO: 66, and the chromosome ChrX-3 site is located by a gRNA shown in SEQ ID NO: 67; the chromosome ChrXIV-Int10 site is located by a gRNA shown in SEQ ID NO: 68, and the chromosome ChrXVI-Int14 site is located by a gRNA shown in SEQ ID NO: 70; the chromosome ChrX-2 site is located by a gRNA shown in SEQ ID NO: 69, and the chromosome ChrII-Int1 site is located by a gRNA shown in SEQ ID NO: 71.

[0080] The nucleotide sequences of each gene, promoter, terminator used in the present application are shown in Table 1:

[0081] Table 1:

[0082]

[0083] Additional aspects and advantages of the present application will be partially given in the following description, partially will become obvious from the following description, or will be understood by the practice of the present application.

[0084] Example 1 Construction of recombinant Saccharomyces cerevisiae strain of 2'-fucosyllactose

[0085] 1.1 Construction of required vector plasmid for the chassis yeast strain

[0086] (1) Plasmid 01 (ChrXII-4-pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1-pTEF2-FutC-tFBA1-pCE-Zero), the role of the plasmid is to knock in the genes of GMER, GMD and FutC at the ChrXII-4 site of Saccharomyces cerevisiae. The relevant characteristics are ΔChrXII-4 pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1-pTEF2-FutC-tFBA1, the GMER gene is controlled by the promoter pHTX7, the GMD gene is controlled by the promoter pPDC1, and the FutC gene is controlled by the promoter pTEF2, the insertion position of the chromosome is ChrXII-4, and the nucleotide sequences of the genes are shown in Table 1.

[0087] The specific construction process is as follows: using the genome of Saccharomyces cerevisiae BY4741 chassis strain as the template, using primers 01-1F and 01-1R in Table 2 to amplify the fragment ChrII-4 left as the homologous left arm of the knock-in template, using primers 01-2F and 01-2R to amplify the promoter fragment pHTX7, primers 01-4F and 01-4R to amplify the terminator fragment tPGK1, primers 01-5F and 01-5R to amplify the terminator fragment tTEF1, primers 01-7F and 01-7R to amplify the promoter fragment pPDC1, primers 01-8F and 01-8R to amplify the promoter fragment pTEF2, primers 01-10F and 01-10R to amplify the terminator fragment tFBA1, and primers 01-11F and 01-11R to amplify the fragment ChrXII-4 as the homologous right arm. Using the synthetic plasmid as the template, using primers 01-3F and 01-3R to amplify the fragment GMER, primers 01-6F and 01-6R to amplify the fragment GMD, and 01-9F and 01-9R to amplify the fragment FutC. The above fragments are recombined and constructed into plasmid 01 by overlap extension PCR method and Novagen One Step Cloning Kit, then amplified in Escherichia coli (Biyun Tian, Catalog No.: D1031S), verified by enzyme digestion, and sequenced correctly to obtain plasmid 01. After linearization by MssI enzyme, the fragment with the target gene is obtained.

[0088] Table 2

[0089]

[0090] The nucleic acid sequences of the promoters are shown in Table 1.

[0091] (2) Plasmid 02 (ChrXI-3 pADH1- Lac12-tCYC1-pCE-Zero)

[0092] The plasmid is used to knock in the gene of Lac12 at the ChrXI-3 site of Saccharomyces cerevisiae. The relevant feature is ΔChrXI-3 pADH1-Lac12-tCYC1, the Lac12 gene is started by using the promoter pADH1, and the insertion position in the chromosome is ChrXI-3.

[0093] The specific construction process is as follows: using the genome of the Saccharomyces cerevisiae BY4741 chassis strain as a template, using primers 02-1F and 02-1R in Table 3 to amplify the fragment ChrXI-3 left as a homologous left arm of the knock-in template, using primers 02-2F and 02-2R to amplify the promoter fragment pADH1, and using primers 02-4F and 02-4R to amplify the terminator fragment tCYC1, using the synthetic plasmid as a template, and using primers 02-3F and 02-3R to amplify the fragment Lac12. The above-mentioned fragments are recombined and constructed into plasmid 02 by overlap extension PCR method and Novizen One Step Cloning Kit, then amplified in Escherichia coli, verified by enzyme digestion, and sequenced correctly to obtain plasmid 02. After linearization by MssI enzyme digestion, the fragment with the target gene is obtained.

[0094] Table 3:

[0095]

[0096] The promoter sequence used is shown in Table 1.

[0097] (3) Plasmid 03 (ChrX-3 pPYK1-CDT2-tADH1-pCE-Zero)

[0098] The plasmid is used to knock in the gene of CDT2 at the ChrX-3 site of Saccharomyces cerevisiae. The relevant feature is ΔChrX-3 pPYK1-CDT2-tADH1, the CDT2 gene is started by using the promoter pPYK1, and the insertion position in the chromosome is ChrX-3.

[0099] The specific construction process is as follows: using the genome of the Saccharomyces cerevisiae BY4741 chassis strain as a template, using primers 03-1F and 03-1R in Table 4 to amplify the fragment ChrX-3 left as a homologous left arm of the knock-in template, using primers 03-2F and 03-2R to amplify the promoter fragment pPYK1, and using primers 03-4F and 03-4R to amplify the terminator fragment tADH1, using the synthetic plasmid as a template, and using primers 03-3F and 03-3R to amplify the fragment CDT2. The above-mentioned fragments are recombined and constructed into plasmid 03 by overlap extension PCR method and Novizen One Step Cloning Kit, then amplified in Escherichia coli, verified by enzyme digestion, and sequenced correctly to obtain plasmid 03. After linearization by MssI enzyme digestion, the fragment with the target gene is obtained.

[0100] Table 4:

[0101]

[0102] The promoter sequences used are shown in Table 1.

[0103] (4) Plasmid 04 (gRNA-ChrXII-4-ChrX-3-ChrXI-3-Cas9)

[0104] The function of the plasmid is to express Cas9 protein and multiple sgRNAs corresponding to three sites ChrXII-4, ChrX-3 and ChrXI-3. The correspondence between each insertion site of the application and gRNA is shown in Table 5.

[0105] Table 5:

[0106]

[0107] Specific construction method: the three DNA fragments containing sgRNA are assembled and constructed by Golden Gate Assembly method provided by pCAS vector, then amplified in E. coli, verified by enzyme digestion and sequenced correctly, and the plasmid 04 is obtained. The nucleotide sequences of each primer used are shown in Table 6.

[0108] Table 6:

[0109]

[0110] 1.2 Construction of chassis yeast strain

[0111] The above plasmids 01, 02 and 03 are linearized by using restriction enzyme MssI, the recovered fragments with target genes and plasmid 04 are co-transformed into Saccharomyces cerevisiae BY4741, and the yeast is genetically edited by LiAc-mediated chemical transformation technology, and the simplified experimental steps are as follows:

[0112] The sample was taken from the frozen bacteria solution, streaked on YPD plate and cultured upside down for 3 days. A single colony was selected and transferred to 3 mL YPD liquid medium, and cultured at 30°C, 220 rpm overnight. The bacteria solution was inoculated into 50 mL YPD medium at an initial OD600 of 0.2, and cultured at 30°C, 220 rpm until the OD600 reached 0.8-1.0. The bacteria solution was centrifuged at 4500 rpm for 5 minutes, and the supernatant was removed and resuspended with 20 mL of 1xTE solution and centrifuged again. 2 mL of solution 1 (1.7 mL ddH2O, 200 μL of 10x lithium acetate, 100 μL of 1xTE) was added, and the solution was incubated at 30°C for 10 minutes to obtain competent cells. 100 μL of competent cells were mixed with 20 μL of ssDNA and 3 μg of DNA fragments, 500 ng of sgRNA, and 600 μL of 50% PEG4000, 75 μL of 1M lithium acetate, and 75 μL of 10xTE were added, and vortexed to mix. The mixture was incubated in a 30°C incubator for 30 minutes, and then 88 μL of DMSO was added and mixed, and the mixture was heated at 42°C for 20 minutes. The mixture was centrifuged at 13000 rpm for 1 minute to remove the supernatant, and 1 mL of YPD liquid medium was added and vortexed to resuspend the bacteria, and the supernatant was removed again. 1 mL of YPD liquid medium was added and vortexed to resuspend the bacteria, and the bacteria were recovered at 30°C, 220 rpm for 60 minutes. The bacteria were centrifuged at 13000 rpm for 1 minute to remove the supernatant, and the bacteria were resuspended with 100 μL of 1xTE, and plated on SD-URA solid plates and cultured for 3-4 days.

[0113] The chromosome multi-site integration was realized by CRISPR-Cas9 gene editing technology, and the marker was uracil. The selected colonies were screened by PCR using genome verification primers XII-4-F / R, XI-3 F / R, and X-3F / R, and a strain with knock-in at all three sites was obtained, named LX-1. The PCR primer sequences used for each colony are shown in Table 7.

[0114] Table 7:

[0115]

[0116] Example 2 Construction of multi-copy number strain

[0117] 2.1 Construction of multi-copy number strain

[0118] The inventors found in previous studies that the key enzymes for biosynthesis of 2'-fucosyllactose are GDP-mannose dehydratase gene (GMD), GDP-L-fucose synthase gene (GMER), and a-(1,2) fucosyltransferase gene (FutC), and the reaction efficiency of the three enzymes affects the yield of 2'-fucosyllactose. In this embodiment, the inventors can affect the expression amount of the enzymes by changing the copy number of the genes corresponding to the three enzymes, so as to regulate the yield of 2'-fucosyllactose.

[0119] Based on LX-1 (i.e., the copy number of GMD, GMER, and FutC genes is 1) obtained in Example 1, five strains with increased copy number of GMD, GMER, and FutC enzymes were obtained by increasing the copy number one by one (the specific copy number is shown in Table 8). The strain construction method is the same as that in Example 1, and the chromosomal region (homologous recombination region) where the gene sequence of the newly introduced enzyme is inserted, the gRNA sequence used, and the colony PCR primer used for screening are shown in the following table.

[0120] Table 8

[0121]

[0122] In other words, the genome of the recombinant microorganism has the following inserted in sequence:

[0123] (A) inserting pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1-pTEF2-FutC-tFBA1 at the Chr XII-4 site of the chromosome, inserting pADH1-Lac12-tCYC1 at the Chr XI-3 site of the chromosome, and inserting pPYK1-CDT2-tADH1 at the Chr X-3 site of the chromosome (the copy number of enzymes in the strain is Lac12, CDT2, GMD, GMER, and FutC);

[0124] (B) inserting pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1-pTEF2-FutC-tFBA1 at the Chr XII-4 site of the chromosome, inserting pADH1-Lac12-tCYC1 at the Chr XI-3 site of the chromosome, inserting pPYK1-CDT2-tADH1 at the Chr X-3 site of the chromosome, and inserting pTEF2-FutC-tFBA1 at the Chr XIV-Int10 site of the chromosome (the copy number of enzymes in the strain is Lac12, CDT2, GMD, GMER, and FutC*2);

[0125] (C) insertion of pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1-pTEF2-FutC-tFBA1 at the Chr XII-4 locus, pADH1-Lac12-tCYC1 at the Chr XI-3 locus, pPYK1-CDT2-tADH1 at the Chr X-3 locus, pTEF2-FutC-tFBA1 at the Chr XIV-Int10 locus, pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1 at the Chr X-2 locus, pTEF2-FutC-tFBA1 at the Chr XVI-Int14 locus (enzyme copy numbers in the strain Lac12, CDT2, GMD*2, GMER*2, FutC*2),

[0126] (D) insertion of pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1-pTEF2-FutC-tFBA1 at the Chr XII-4 locus, pADH1-Lac12-tCYC1 at the Chr XI-3 locus, pPYK1-CDT2-tADH1 at the Chr X-3 locus, pTEF2-FutC-tFBA1 at the Chr XIV-Int10 locus, pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1 at the Chr X-2 locus, pTEF2-FutC-tFBA1 at the Chr XVI-Int14 locus (enzyme copy numbers in the strain Lac12, CDT2, GMD*2, GMER*2, FutC*3),

[0127] (E) insertion of pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1-pTEF2-FutC-tFBA1 at the Chr XII-4 locus, pADH1-Lac12-tCYC1 at the Chr XI-3 locus, pPYK1-CDT2-tADH1 at the Chr X-3 locus, pTEF2-FutC-tFBA1 at the Chr XIV-Int10 locus, pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1 at the Chr X-2 locus, pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1 at the Chr II-Int1 locus (enzyme copy numbers in the strain Lac12, CDT2, GMD*3, GMER*3, FutC*2); and

[0128] (F) insertion of pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1-pTEF2-FutC-tFBA1 at the Chr XII-4 locus, insertion of pADH1-Lac12-tCYC1 at the Chr XI-3 locus, insertion of pPYK1-CDT2-tADH1 at the Chr X-3 locus, insertion of pTEF2-FutC-tFBA1 at the Chr XIV-Int10 locus, insertion of pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1 at the Chr X-2 locus, insertion of pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1 at the Chr II-Int1 locus, insertion of pTEF2-FutC-tFBA1 at the Chr XVI-Int14 locus (enzyme copy numbers in the strain Lac12, CDT2, GMD*3, GMER*3, FutC*3).

[0129] 2.2 Shake flask fermentation of S. cerevisiae strains and detection of 2'-fucosyllactose production

[0130] The part of the experiment is aimed at the various 2'-fucosyllactose recombinant yeast strains constructed in this embodiment, a set of shake flask fermentation method is developed. The specific parameters and process are as follows: take-80℃ frozen each gene copy number different Lac12, CDT2, GMD, GMER, FutC group, Lac12, CDT2, GMD, GMER, FutC*2 group, Lac12, CDT2, GMD*2, GMER*2, FutC*2 group, Lac12, CDT2, GMD*2, GMER*2, FutC*3 group, Lac12, CDT2, GMD*3, GMER*3, FutC*2 group, Lac12, CDT2, GMD*3, GMER*3, FutC*3 group strain, pick 8 single clones on the configured YPDA plate (YPDA plate: 1% yeast extract, peptone, 2% glucose, 2% agar powder, 115 degree high pressure sterilization for 15 minutes); after growing for 48 hours, pick single clones (3*8) into 50 mL centrifuge tube, use 5 mL SD culture based on 30℃ culture for 24 hours; transfer to OD=0.1 (60µL dilution to 5mL medium), 5mL of SD medium, test OD every 1 hour (a total of 5 hours); continue to culture for about 13 hours, select the normal growth rate (70-90 minutes a generation) strain 250µL inoculated into 50mL of SD medium (250mL flask) at 30℃ (inoculation amount is 0.5%, the upper tank fermentation should be increased to 5%); after 12 hours of culture, measure OD, when OD600 =1~3, add lactose uniformly, and the final concentration is 0.44% (1mL concentration is 220g / L mother liquor added to 50 mL medium); continue to culture at 30℃ for T = 24 hours, then take sample; take 5mL, measure its OD600 value. After fermentation, the sample is detected for 2'-fucosyllactose yield. The SD medium used in this embodiment is configured by using SC comolete medium of YGM003A-1 of Panji Nuo Company and 2% glucose, and then high pressure sterilization at 115℃ for 15 minutes.

[0131] The yield of 2'-fucosyllactose of the strains constructed in each group is as follows Figure 5As shown, in the case of copy number increase, the yield of 2'-fucosyllactose is significantly improved, but it is not that the more the copy number, the higher the yield. In this embodiment, when the copy number of the strain genes is Lac12, CDT2, GMD*3, GMER*3, FutC*2, the yield is the highest, and the yield of 2'-fucosyllactose is more than 2.5 times higher than that of the strain of the Lac12, CDT2, GMD, GMER, FutC group. That is, when the GMD and GMER enzymes are 3 copies, the yield of FutC enzyme with 2 copies is higher than that with 3 copies, which indicates that copy number optimization is not a linear process, often involving the reaction rate of substrates and enzymes in the pathway, and the dynamic change process of concentration. The yield improvement obtained by adjusting the copy number by the present technology cannot be predicted by simple parameter simulation or calculation, but can provide supportive understanding and data for related calculations. While obtaining high-yield strains, new understanding of the influence of copy number on 2-FL synthesis process is provided.

[0132] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0133] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A recombinant microorganism, characterized in that, The recombinant microorganism carries nucleic acids encoding GDP-mannose dehydratase, GDP-L-fucose synthase, alpha-(1,2)fucosyltransferase, lactose transporter and oligosaccharide transporter protein, wherein: the nucleotide sequence encoding the GDP-mannose dehydratase is shown as SEQ ID NO: 1, the nucleotide sequence encoding the GDP-L-fucose synthase is shown as SEQ ID NO: 2, the nucleotide sequence encoding the alpha-(1,2)fucosyltransferase is shown as SEQ ID NO: 3, the nucleotide sequence encoding the lactose transporter is shown as SEQ ID NO: 4, the nucleotide sequence encoding the oligosaccharide transporter protein is shown as SEQ ID NO: 5, the copy number of the nucleic acid encoding the GDP-mannose dehydratase is 3, the copy number of the nucleic acid encoding the GDP-L-fucose synthase is 3, the copy number of the nucleic acid encoding the alpha-(1,2)fucosyltransferase is 2, the copy number of the nucleic acid encoding the lactose transporter is 1, the copy number of the nucleic acid encoding the oligosaccharide transporter protein is 1, inserting pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1-pTEF2-FutC-tFBA1 at the Chr XII-4 locus, inserting pADH1-Lac12-tCYC1 at the Chr XI-3 locus, inserting pPYK1-CDT2-tADH1 at the Chr X-3 locus, inserting pTEF2-FutC-tFBA1 at the Chr XIV-Int10 locus, inserting pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1 at the Chr X-2 locus, inserting pHXT7-GMER-tPGK1-pPDC1-GMD-tTEF1 at the Chr II-Int1 locus, the recombinant microorganism takes Saccharomyces cerevisiae as a chassis strain.

2. The recombinant microorganism of claim 1, wherein, the nucleic acid sequence encoding the GDP-mannose dehydratase and the nucleic acid sequence encoding the GDP-L-fucose synthase are derived from Escherichia coli, and / or the nucleic acid sequence encoding the alpha-(1,2)fucosyltransferase is derived from Helicobacter pylori, and / or the nucleic acid sequence encoding the lactose transporter is derived from Kluyveromyces lactis, and / or the nucleic acid sequence encoding the oligosaccharide transporter protein is derived from Neurospora crassa.

3. Use of the recombinant microorganism of any one of claims 1-2 in the preparation of 2'-fucosyllactose.

4. A method of preparing 2'-fucosyllactose, characterized in that, comprising: liquid culturing the recombinant microorganism of any one of claims 1-2 inoculated into a liquid medium; and isolating 2'-fucosyllactose from the culture product of the liquid culturing.

5. The method of claim 4, wherein, the inoculation amount of the recombinant microorganism in the inoculation process is 0.5%-5%; and / or the culturing comprises: when the recombinant microorganism is cultured to an OD600 value of 1-3, adding lactose, and the final concentration of lactose in the liquid medium is 0.40%-0.50%.

6. The method of claim 5, wherein, After the addition of lactose, the recombinant microorganism is further cultured for 20-28 hours to obtain 2'-fucosyllactose, and / or the culturing is performed at 28-32 DEG C.

7. The method of claim 4, wherein, The liquid culture medium comprises a base culture medium and glucose, wherein the final concentration of glucose is 2-4%, and / or the base culture medium is an SD culture medium.

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