Engineered bacteria for preparing human milk oligosaccharides without by-products and application thereof

Abstract

The application provides an engineering bacterium for preparing human milk oligosaccharide without by-products and an application thereof, and relates to the technical field of genetic engineering. In order to solve the problem that the proportion of by-product LNTri II is high and the by-product is difficult to purify when LNT or LNnT is produced by microbial fermentation, on the basis of an LNT or LNnT production strain, a new engineering bacterium for producing lactose-N-tetrasaccharide or lactose-N-neotetrasaccharide is obtained by integrating a temperature-controlled promoter and beta-acetylhexosaminidase. The expression of BbhI is controlled by a temperature-controlled promoter for the first time, so that the problem of yield reduction caused by the continuous expression of BbhI in the engineering strain in the prior art is solved. By screening the suitable fermentation OD 600 of the engineering bacterium and temperature, the purpose of eliminating the by-product LNTri II in the fermentation liquor while improving the yield of LNT or LNnT is achieved, the downstream purification step is saved, the production cost is effectively reduced, and the engineering bacterium has a wide industrialization prospect.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, and in particular to an engineered bacterium for preparing human milk oligosaccharides without byproducts and its applications. Background Technology

[0002] Human milk oligosaccharides (HMOs) are the third most abundant solid component in breast milk after lactose and lipids. Considered natural prebiotics for infants, they can shape the gut microbiota, promote gut health, and help safeguard infant health, including promoting brain and immune system development. They also exhibit anti-adhesion and antibacterial activity, reducing the incidence of enteritis. Therefore, the structure, physiological functions, and synthesis methods of HMOs have received widespread attention in recent years. Adding HMOs to infant formula has become a future development trend. HMOs are a collective term for soluble oligosaccharides found in human milk, among which lactose-N-tetrasaccharide (LNT) and lactose-N-neotetrasaccharide (LNnT) have been approved by the U.S. Food and Drug Administration (FDA) for commercial use. Several brands have already added LNT and LNnT to commercial infant formula. For example, LNT is added to Nestlé BEBA Supreme at concentrations of 30, 10, and 9 mg / 100 mL for stages 1, 2, and 3; LNnT is added to Wyeth's Illuma formula at concentrations of 0.5, 0.13, 0.11, and 0.11 g / L for stages 1, 2, 3, and 4.

[0003] Both LNT and LNnT are composed of galactose, N-acetylglucosamine, and glucose. The structure of LNT is Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc, and the structure of LNnT is Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc. Currently, the main synthetic methods for LNT and LNnT include chemical synthesis, in vitro enzymatic synthesis, and microbial fermentation. Chemical synthesis involves complex reactions, expensive raw materials, and high production costs, making it unsuitable for industrial production. In vitro enzymatic synthesis requires nucleotide sugars and cofactors as substrates, which are expensive, and the low enzyme conversion rate also makes it unsuitable for large-scale industrial production. Microbial fermentation, on the other hand, can use inexpensive carbon sources as substrates and meets the requirements of environmental protection and sustainable development, thus attracting considerable attention from researchers in recent years.

[0004] In 2014, Baumgärtner et al. first developed an engineered strain that synthesized LNTs using Escherichia coli as a host, by knocking out the gene encoding β-galactosidase. lacZA bacterial β-1,3-acetylglucosamine transferase (LgtA, EC 2.4.1.149) was introduced from Neisseria meningitidis to synthesize LNTri II (GlcNAcβ1-3Galβ1-4Glc), an important precursor for the synthesis of LNT and LNnT. Based on this, a β-1,3-galactosyltransferase WbgO from Escherichia coli O55:H7 was introduced to catalyze the galactosylation of LNTri II to produce LNT; conversely, the introduction of a β-1,4-galactosyltransferase LgtB catalyzes the galactosylation of LNTri II to produce LNnT.

[0005] LNTri II, a precursor to LNT / LNnT, is released extracellularly via transport proteins during production and accumulates as a byproduct, with the LNTri II to LNT / LNnT ratio reaching as high as 1:2. Excessive accumulation of LNTri II leads to reduced yield of the target product LNT / LNnT and increased difficulty in downstream separation and purification. A 2023 article (https: / / doi.org / 10.1021 / acs.jafc.3c03644) discusses... bbhI The vectors pACYCDuet-1, pCOLADuet-1, and pCDFDuet-1 were cloned and introduced into engineered strains. BbhI successfully degraded LNTri II in the engineered strains, with almost no residual LNTri II detected, but the yield of LNT was also significantly reduced. Summary of the Invention

[0006] This invention addresses the problem of high LNTri II content and difficulty in purification as a byproduct in existing microbial fermentation production of LNT or LNnT. Based on LNT or LNnT production strains MG07 or MG11 constructed using *E. coli* as a chassis, the temperature-controlled promoter CI857-pRpL and the β-acetylhexosaminease gene are incorporated... bbhI The expression cassette was integrated into the genome of the aforementioned production strain to obtain production strains MG08 or MG12. Production strains MG08 or MG12 were then processed by controlling fermentation OD... 600 Temperature control was used to eliminate the byproduct LNTri II in the fermentation broth while increasing the yield of LNT / LNnT.

[0007] On the one hand, this application provides engineered bacteria that produce lactose-N-tetrasaccharide and / or lactose-N-neotetrasaccharide, said engineered bacteria expressing a temperature-controlled promoter and β-acetylhexosaminease.

[0008] Preferably, the temperature-controlled promoter can control the expression of β-acetylhexosaminease by regulating the temperature.

[0009] Preferably, the temperature-controlled starter is activated when the temperature is >30°C.

[0010] Further, the temperature-controlled promoter includes CI857-pRpL; preferably, the temperature-controlled promoter includes the sequence shown in SEQ ID No. 12 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID No. 12.

[0011] Further, the β-acetylhexosamine enzyme comprises the sequence shown in SEQ ID No. 14 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID No. 14.

[0012] Preferably, the nucleotide sequence encoding the β-acetylhexosamine enzyme comprises the sequence shown in SEQ ID No. 13 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID No. 13.

[0013] Furthermore, the engineered bacterial genome integrates and expresses a temperature-controlled promoter and β-acetylhexosaminease.

[0014] Preferably, the integration site can be selected from... rpH , ybeQ , ldhA , wecB、manY、manZ、manX、nagB、poxB、 ygaY、arsB One or more sites.

[0015] Furthermore, the engineered bacteria are selected from one or more of Escherichia coli, Streptococcus, Bacillus, Saccharomyces cerevisiae, and Pichia pastoris.

[0016] Preferably, the engineered bacteria is Escherichia coli.

[0017] Furthermore, the engineered bacteria can increase the yield of lactose-N-tetrasaccharide and / or lactose-N-neotetrasaccharide while eliminating lactose-N-trisaccharide (LNTri II).

[0018] In a preferred embodiment, the *E. coli* may be... E. coli K12 MG1655, E. coli BL21 (DE3) E. coli One or more of DH5α.

[0019] Those skilled in the art will understand that conventional fermentation strains or any known industrial strain can also be used as the starting strain, and no specific strain is limited here.

[0020] Furthermore, the engineered bacteria also include any one or more of the following modifications: A1) Low or no expression of β-galactosidase (lacZ), Escherichia coli membrane polysaccharide synthesis-related genes (wcaJ), and / or UDP-glucose dehydrogenase (ugd). A2) Expression or overexpression of UDP-galactose-4-epimerase (galE), UDP-glucose pyrophosphorylase (galU), galactosyltransferase and / or β-1,3-N-acetylglucosamine transferase (lgtA).

[0021] Preferably, the low expression or non-expression is achieved by knocking out the gene corresponding to the enzyme.

[0022] Preferably, the expression or overexpression is achieved by integrating at least one copy of the gene corresponding to the enzyme.

[0023] Those skilled in the art can also achieve low expression or no expression, or expression or overexpression, through known methods, as long as the effect can be achieved, and this application does not make specific limitations on this.

[0024] Those skilled in the art can select appropriate integration sites according to actual needs.

[0025] Preferably, the integration site can be selected from... rpH , ybeQ , ldhA , wecB、manY、manZ、manX、nagB、poxB、 ygaY、arsB One or more sites.

[0026] More preferably, the copy number and its upper and lower limits can be any value of 1, 2, 3, 4, or 5.

[0027] Furthermore, the galactosyltransferase includes β-1,3-galactosyltransferase (wbgO) and / or β-1,4-galactosyltransferase (HP3).

[0028] Furthermore, the copy number ratio of UDP-galactose-4-epimerase (galE), UDP-glucose pyrophosphorylase (galU), galactosyltransferase, and β-1,3-N-acetylglucosamine transferase (lgtA) is (1-5):(1-5):(1-5):(1-5).

[0029] Preferably, the copy number ratio of UDP-galactose-4-epimerase (galE), UDP-glucose pyrophosphorylase (galU), galactosyltransferase, and β-1,3-N-acetylglucosamine transferase (lgtA) is 1:1:4:3.

[0030] It is understood that those skilled in the art can choose engineered bacteria that can produce lactose-N-tetrasaccharide or lactose-N-neotetrasaccharide according to the actual situation, and no further limitations are made here.

[0031] In a preferred embodiment, the engineered bacteria producing lactose-N-tetrasaccharide comprises the following modifications: low or no expression of β-galactosidase (lacZ), *E. coli* membrane polysaccharide synthesis-related genes (wcaJ), and / or UDP-glucose dehydrogenase (ugd); expression or overexpression of UDP-galactose-4-epimerase (galE), UDP-glucose pyrophosphorylase (galU), and β-1,3-galactosyltransferase genes (…). wbgO ) and / or β-1,3-N-acetylglucosamine transferase (lgtA); temperature-controlled promoters control β-acetylglucosamine enzyme expression.

[0032] Preferably, the engineered bacteria producing lactose-N-tetrasaccharide include the following modifications: low expression or non-expression of β-galactosidase (lacZ), the *E. coli* membrane polysaccharide synthesis-related gene (wcaJ), and / or UDP-glucose dehydrogenase (ugd); rpH Site integration of UDP-galactose-4-epimerase (galE), in ybeQ Site integration of UDP-glucose pyrophosphorylase (galU), in ldhA , wecB、manY、manZ Four copies of the β-1,3-galactosyltransferase gene were integrated at the site. wbgO ) and / or nagB、poxB、ygaY Three copies of β-1,3-N-acetylglucosamine transferase (lgtA) are integrated at the site; arsB The site integrates a temperature-controlled promoter and β-acetylhexosaminease, with the temperature-controlled promoter controlling the expression of β-acetylhexosaminease.

[0033] In a preferred embodiment, the engineered bacteria producing lactose-N-neotetrasaccharide are modified as follows: low or no expression of β-galactosidase (lacZ), *E. coli* membrane polysaccharide synthesis-related genes (wcaJ), and / or UDP-glucose dehydrogenase (ugd); and expression or overexpression of UDP-galactose-4-epimerase (galE), UDP-glucose pyrophosphorylase (galU), and β-1,4-galactosyltransferase genes. (HP3) And / or β-1,3-N-acetylglucosamine transferase (lgtA); temperature-controlled promoters control β-acetylglucosamine expression.

[0034] Preferably, the engineered bacteria producing lactose-N-neotetrasaccharide include the following modifications: low expression or non-expression of β-galactosidase (lacZ), the *E. coli* membrane polysaccharide synthesis-related gene (wcaJ), and / or UDP-glucose dehydrogenase (ugd); rpH Site integration of UDP-galactose-4-epimerase (galE), in ybeQ Site integration of UDP-glucose pyrophosphorylase (galU), in ldhA、wecB、manX、manZ β-1,4-galactosyltransferase gene with 4 copies at site 4 (HP3) and / or nagB、poxB、ygaY Three copies of β-1,3-N-acetylglucosamine transferase (lgtA) are integrated at the site; arsB The site integrates a temperature-controlled promoter and β-acetylhexosaminease, with the temperature-controlled promoter controlling the expression of β-acetylhexosaminease.

[0035] It is understandable that those skilled in the art can select appropriate gene editing systems and methods to construct the aforementioned engineered bacteria based on the actual situation.

[0036] On the other hand, this application also provides biological materials, said biological materials comprising any one of the following B1)-B3): B1) Expression cassette, which expresses a temperature-controlled promoter and β-acetylhexosaminease; B2) A recombinant vector containing the expression cassette described in B1); B3) Whole-cell catalyst, wherein the whole-cell catalyst contains the expression cassette of B1), the recombinant vector of B2) and / or the engineered bacteria.

[0037] Preferably, the temperature-controlled starter includes CI857-pRpL.

[0038] Those skilled in the art will recognize that other commonly used expression elements can be adaptively added to the expression cassette described in this application to assist in the expression of the target gene. In a preferred embodiment, the expression cassette is a CI-bbhI expression cassette, which includes CI-PRL (CI857-pRpL, a temperature-controlled promoter) and the BbhI gene, and the CI-bbhI nucleotide sequence is shown in SEQ ID No. 11.

[0039] Recombinant vectors can be any vector (e.g., plasmids or viruses) that facilitates the manipulation of recombinant DNA and the expression of nucleic acid sequences. The choice of vector typically depends on its compatibility with the host cell into which it will be introduced. Vectors can be linear or closed circular plasmids. Vectors can be self-replicating vectors (i.e., complete structures existing outside the chromosome that can replicate independently of the chromosome), such as plasmids, extrachromosomal elements, minichromosomes, or artificial chromosomes. Vectors can contain any mechanism that ensures self-replication. Alternatively, a vector is a vector that, when introduced into a host cell, will integrate into the genome and replicate along with the integrated chromosome. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, can be used, collectively containing the entire DNA to be introduced into the host cell's genome.

[0040] In a preferred embodiment, the recombinant vector may be pETDuet-1.

[0041] On the other hand, this application also provides a method for preparing lactose-N-tetrasaccharide and / or lactose-N-neotetrasaccharide, the method comprising: culturing the engineered bacteria.

[0042] Furthermore, the culture temperature is 37℃-42℃.

[0043] The value of the culture temperature and the upper or lower limit of the culture temperature range can be any value among 37℃, 38℃, 39℃, 40℃, 41℃, and 42℃.

[0044] Furthermore, the cultured OD 600 When the value is 15-40, expression of β-acetylhexosaminease ceases.

[0045] The cultured OD 600 Values ​​and OD 600 The upper or lower limit of the value range can be any value from 15, 15.6, 15.8, 16, 17, 18, 19, 19.2, 19.5, 20, 21, 22, 23, 24, 25, 26, 26.9, 27, 27.1, 28, 29, 29.1, 29.8, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40.

[0046] Preferably, the cultured OD 600 When the value is 15-25, expression of β-acetylhexosaminease ceases.

[0047] Preferably, the cessation of β-acetylhexosamine enzyme expression is achieved by controlling the culture temperature, thereby controlling the temperature-controlled promoter.

[0048] More preferably, the cessation of β-acetylhexosamine expression is achieved by lowering the culture temperature; more preferably, the culture temperature is lowered to ≤30°C.

[0049] In a preferred embodiment, a method for preparing lactose-N-tetrasaccharide and / or lactose-N-neotetrasaccharide, the method comprising: Step 1: Construct the engineered bacteria; Step 2: Incubate the engineered bacteria at 37℃-42℃ to cultivate OD. 600 When the value is 15-40, expression of β-acetylhexosaminease ceases.

[0050] Preferably, the method includes: Step 1: Construct the engineered bacteria; Step 2: Incubate the engineered bacteria at 37℃-42℃ to cultivate OD. 600 When the value is 15-40, stop expressing β-acetylhexosaminease, add D-lactose, and after the glycerol in the culture medium is consumed, add glycerol and ferment.

[0051] More preferably, the method includes: Step 1: Construct the engineered bacteria; Step 2: Inoculate the engineered bacteria into an activation medium for activation until OD... 600 =1.2-1.5, to obtain seed culture medium; Step 3: Inoculate the seed culture medium into the fermenter at an inoculum rate of 1-3% (v / v). Initial culture conditions are: culture temperature 37℃-42℃, pH: 6.7-6.9, rotation speed 10-100 rpm, aeration rate: 0.8-1.2 vvm, and culture OD... 600 When the value is 15-40, cool down to 30℃ to stop the expression of β-acetylhexosamine enzyme, add D-lactose, and control the residual amount <2 g / L. During fermentation, control the dissolved oxygen at 20-30%. After the glycerol in the culture medium is almost consumed, add glycerol, and control the residual amount <1 g / L. Ferment for 70-100 h or until the product no longer increases significantly.

[0052] In this application, a suitable temperature range was obtained through screening experiments that did not affect the growth of the strain or the leakage-induced expression of BbhI by the temperature-controlled promoter. Furthermore, the fermentation yield of the engineered strain was improved and the byproduct LNTri II was eliminated by optimizing the expression intensity of BbhI.

[0053] Preferably, the product obtained by the method does not contain byproducts, the byproducts including LNTri II.

[0054] On the other hand, this application also provides the use of the engineered bacteria or the biological material in any one or more of the following C1)-C5): C1) Production of lactose-N-tetrasaccharide and / or lactose-N-neotetrasaccharide; C2) Increase the yield of lactose-N-tetrasaccharide and / or lactose-N-neotetrasaccharide; C3) Improve the purity of lactose-N-tetrasaccharide and / or lactose-N-neotetrasaccharide; C4) Reduces the content of the byproduct lactose-N-trisaccharide (LNTri II); C5) Increases the yield of lactose-N-tetrasaccharide and / or lactose-N-neotetrasaccharide while eliminating lactose-N-trisaccharide (LNTriII).

[0055] Preferably, the yield of lactose-N-tetrasaccharide is ≥86 g / L, and the yield of lactose-N-neotetrasaccharide is ≥24 g / L.

[0056] In this application, a temperature-controlled promoter is used to control the expression intensity and duration of β-acetylhexosaminease. This effectively reduces or eliminates the byproduct LNTri II while increasing the yield and purity of lactose-N-tetrasaccharide and / or lactose-N-neotetrasaccharide products. It effectively controls metabolic flux, ensures that the substrate is converted into the product to the maximum extent during the synthesis process, improves production efficiency, and is beneficial to the purification and production of the target product.

[0057] The present invention has the following beneficial effects: This application addresses the problem of high proportion and difficulty in purification of the byproduct LNTri II during existing microbial fermentation production of LNT or LNnT without affecting LNT or LNnT yield. Based on LNT or LNnT production strains MG07 or MG11 constructed using Escherichia coli as a chassis, this application integrates the temperature-controlled promoter CI857-pRpL and the β-acetylhexosaminease gene. bbhI The expression cassette yielded new engineered bacteria MG08 or MG12 producing lactose-N-tetrasaccharides or lactose-N-neotetrasaccharides. For the first time, BbhI expression was controlled via a temperature-controlled promoter, solving the problem of reduced yield caused by continuous BbhI expression in engineered strains in existing technologies.

[0058] This application also provides a novel method for preparing lactose-N-tetrasaccharide or lactose-N-neotetrasaccharide by fermentation using engineered bacteria, by screening engineered bacteria MG08 or MG12 for suitable fermentation OD. 600By adjusting the temperature, the goal of eliminating the byproduct LNTri II in the fermentation broth while increasing the yield of LNT or LNnT was achieved, saving downstream purification steps, effectively reducing production costs, and showing broad prospects for industrialization. Attached Figure Description

[0059] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 for CI-bbhI Plasmid mapping; Figure 2 Statistical chart of LNT production results by dual fermentation of MG08 and BL21(DE3) with pETDuet-1-bbhI; Figure 3 Statistical chart of LNnT production results by dual fermentation of MG11 and BL21(DE3) and pETDuet-1-bbhI. Detailed Implementation

[0060] Technical terms: Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution studies.

[0061] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.

[0062] Expression cassette: An expression cassette is a set of DNA sequences that consists of promoters, target genes, and reporter genes, and can be expressed in specific tissues and is easily detected.

[0063] Recombinant vectors: Recombinant vectors are vectors into which the target gene is transferred based on the basic framework of a cloning vector, thereby enabling the target gene to be expressed.

[0064] Recombinant microorganisms (engineered bacteria): bacterial cell lines that use genetic engineering methods to achieve efficient expression of exogenous genes.

[0065] Recombinant cells: The term "recombinant cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "recombinant cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0066] Whole-cell catalysts: Whole-cell biocatalysis refers to the process of using a complete biological organism (i.e., whole cell, tissue, or even individual) as a catalyst for chemical transformation. The complete biological organism that participates in this catalytic process is called a whole-cell catalyst.

[0067] Free expression: Free expression is the expression of target genes using free plasmids. Free plasmids are independent DNA molecules that exist in cells and have the ability to replicate and be transmitted independently. They are widely used in genetic engineering and molecular biology research.

[0068] Integrated expression: Expression that occurs when a gene is integrated into the genome.

[0069] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0070] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0071] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0072] Unless otherwise specified, all reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are commercially available products. Where specific conditions are not specified in the embodiments, they are performed under standard conditions or conditions recommended by the manufacturer.

[0073] The plasmids, restriction enzymes, PCR enzymes, column DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products. The specific operations were performed according to the kit instructions.

[0074] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, microbiology, biochemistry, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Specifically, they can be performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0075] In addition, the term "water" in this invention includes any feasible type of water that can be used in the art, such as purified water, deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and pure water.

[0076] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0077] The reagents involved in the following examples are as follows: 2×Hieff PCR Master mix was purchased from Yisheng Biotechnology, 2×High fidelity Master mix was purchased from MCLAB, plasmid miniprep kit was purchased from Omega, universal DNA purification and recovery kit was purchased from Tiangen, DpnⅠ was purchased from Thermo, and 2×Seamless Cloning Mix was purchased from Biomed.

[0078] The strains, plasmids, culture media, and feeds involved in the following examples are as follows: (1) Strains: E. coli K12 MG1655, E. coli BL21 (DE3) E. coli DH5α competent cells were purchased from Beijing TransGen Biotech Co., Ltd. (2) Plasmids: pTargetF and pCpf1 were purchased from Addgene; (3) Culture medium and feed: a. LB medium formula (20 g / L agar powder added to LB solid medium) Table 1 LB medium formulation

[0079] b. DM culture medium formulation Table 2 DM medium formulation

[0080] c. Trace element solution Table 3. Trace Element Solution Formula

[0081] d. Preparation and sterilization of culture media and feed additives Solid medium (LB): Prepare 100 mL of LB medium according to the LB medium formula (Table 1), at natural pH, and pour it into an eggplant flask or shake flask. Add agar powder according to the ratio, sterilize the prepared medium at 121°C for 20 min, and then cool it.

[0082] Shake flask seed culture medium (LB): Prepare 400 mL of LB medium according to the LB medium formula (Table 1), at natural pH, and fill 4-5 500 mL shake flasks, each with a volume of 80 mL. Sterilize the filled medium at 121°C for 20 min, and then cool.

[0083] Fermentation medium (DM): Prepare the fermentation medium according to the formula in Table 2, with an initial fermentation volume of 2 L. Accurately weigh each raw material and put it into the fermenter, stir and dissolve thoroughly. Add 1 ml / L of defoamer before sterilization, sterilize at 121℃ for 30 min, and then cool to 37℃. After sterilization, add ammonium sulfate hydrochloride and adjust the pH to 6.8 before use.

[0084] Carbon source replenishment bottle (600 g / L): Accurately weigh 3 kg of glycerol and 100 g of magnesium sulfate heptahydrate, dissolve them in an appropriate amount of purified water, prepare 5 L of solution, sterilize at 115℃ for 30 min, and store at room temperature. Add 5 mL of 200 g / L sterile ammonium sulfate hydrochloride to the sterilized and cooled mixed solution of glycerol and magnesium sulfate heptahydrate, to make the final concentration of ammonium sulfate hydrochloride 0.2 g / L, and store at room temperature.

[0085] 200 g / L ammonium sulfate hydrochloride solution: Accurately weigh 120 g of ammonium sulfate hydrochloride, add an appropriate amount of sterilized purified water to dissolve it, and then dilute to volume with a 600 mL volumetric flask to prepare a 200 g / L ammonium sulfate hydrochloride solution. Filter and sterilize in a clean bench and store at -20℃ protected from light.

[0086] Lactose replenishment bottle (200 g / L): Accurately weigh 200 g of lactose, dissolve it in an appropriate amount of purified water to prepare 1 L of 200 g / L lactose solution, sterilize at 115℃ for 30 min, and store at room temperature. Separately weigh 1 kg of lactose, dissolve it in an appropriate amount of purified water to prepare 5 L of 200 g / L lactose solution, sterilize at 115℃ for 30 min, and store at room temperature for later use.

[0087] Ammonia solution: Use analytical grade ammonia solution.

[0088] The concentrations of kanamycin, spectinomycin hydrochloride, spectinomycin, and ampicillin in the culture medium were 100 μM / L.

[0089] The methods for preparing competent states mentioned below include: Preparation of competent cells of strain K12 MG1655: Single colonies of K12 MG1655 or K12 MG1655 strain preserved in glycerol were inoculated into 4 mL of antibiotic-free LB medium and cultured overnight at 37°C and 220 rpm to obtain seed culture. The seed culture was then inoculated into antibiotic-free LB medium at a rate of 1% and cultured at 37°C and 220 rpm until OD was reached. 600 =0.6-0.8. After incubating the bacterial culture on ice for 30 min, centrifuge at 5000 rpm for 5 min at 4℃, and discard the supernatant. Resuspend the bacterial pellet in 20 mL of 10% glycerol and centrifuge again at 5000 rpm for 5 min at 4℃. Repeat this step three times. Finally, resuspend the bacterial pellet in 500 μL of 10% glycerol, aliquot into 10 tubes, and store at -80℃.

[0090] The pCpf1 plasmid was electroporated into competent cells of K12MG1655 strain to obtain K12MG1655 strain containing the pCpf1 plasmid.

[0091] Preparation of competent cells of K12 MG1655 (K12 MG1655+pCpf1) strain containing pCpf1 plasmid: Single colonies of K12 MG1655 containing the pCpf1 plasmid or K12 MG1655 strain containing the pCpf1 plasmid preserved in glycerol were inoculated into 4 mL of antibiotic-free LB medium and cultured overnight at 37°C and 220 rpm to obtain seed culture. The seed culture was then inoculated at a 1% inoculation rate into LB medium containing kanamycin at 37°C and 220 rpm until OD was reached. 600 When the OD value is 0.1-0.2, add 10 M mL L-arabinose for induction, and continue culturing at 30℃ and 220 rpm until OD reaches 0.2. 600 =0.6-0.8. After incubating the bacterial culture on ice for 30 min, centrifuge at 5000 rpm for 5 min at 4℃, and discard the supernatant. Resuspend the bacterial pellet in 20 mL of 10% glycerol and centrifuge again at 5000 rpm for 5 min at 4℃. Repeat this step three times. Resuspend the bacterial pellet in 500 μL of 10% glycerol, aliquot into 10 tubes, and store at -80℃.

[0092] The competent cells of the K12 MG1655 (K12 MG1655+pCpf1) strain containing the pCpf1 plasmid can be competent cells of strains MG01 / MG02 / MG03 / MG04 / MG07 / MG011.

[0093] The following genes are involved: lacZ Nucleotide sequences are as shown in SEQ ID No. 1, and amino acid sequences are as shown in SEQ ID No. 2; wcaJ Nucleotide sequences are as shown in SEQ ID No. 3, and amino acid sequences are as shown in SEQ ID No. 4; ugd Nucleotide sequences are as shown in SEQ ID No. 5, and amino acid sequences are as shown in SEQ ID No. 6; lgtA Nucleotide sequences are as shown in SEQ ID No. 7, and amino acid sequences are as shown in SEQ ID No. 8; wbgO Nucleotide sequences are shown in SEQ ID No. 9, and amino acid sequences are shown in SEQ ID No. 10; CI-bbhI nucleotide sequences are shown in SEQ ID No. 11, wherein CI-PRL (temperature-controlled promoter) nucleotide sequences are shown in SEQ ID No. 12, BbhI nucleotide sequences are shown in SEQ ID No. 13, and BbhI amino acid sequences are shown in SEQ ID No. 14; galE Nucleotide sequences are as shown in SEQ ID No. 15, and amino acid sequences are as shown in SEQ ID No. 16; galU Nucleotide sequences are as shown in SEQ ID No. 17, and amino acid sequences are as shown in SEQ ID No. 18; HP3 Nucleotide sequences are shown in SEQ ID No. 19, and amino acid sequences are shown in SEQ ID No. 20.

[0094] rpH The GenBank Accession Number for this site is WP_219192980.1. ybeQ The GenBank Accession Number for this site is WP_274486148.1. ldhA The GenBank Accession Number for this site is EPC8574109.1. wecB The GenBank Accession Number for this site is WP_375156771.1. manY The GenBank Accession Number for this site is WP_097343425.1. manZ The GenBank Accession Number for this site is WP_208488765.1. manX The GenBank Accession Number for this site is MCK3022992.1. nagB The GenBank accession number for this site is WP_079985210.1. poxB The GenBank Accession Number for this site is WP_449401977.1. ygaYThe GenBank Accession Number for this site is CP025268.1. arsB The GenBank Accession Number for this site is WP_454081593.1.

[0095] Example 1 Construction of LNT / LNnT engineered strain This embodiment provides the construction of LNT / LNnT engineered strains, including the following steps: (1) Using wild-type Escherichia coli K12 MG1655 as the host, the gene encoding β-galactosidase was knocked out. lacZ Escherichia coli membrane polysaccharide synthesis-related genes wcaJ and UDP-glucose dehydrogenase gene ugd Obtain K12 MG1655Δ lacZ Δ wcaJ Δ ugd The strain was named MG01; (2) Based on MG01 rpH The site integrates 1 copy number of galE ,exist ybeQ The site integrates 1 copy number of galU strain K12 MG1655Δ was obtained. lacZ Δ wcaJ Δ ugd Δ rpH :: galE Δ ybeQ :: galU It was named MG02.

[0096] (3) Based on MG02, respectively ldhA , wecB、manY、manZ Four copies of the β-1,3-galactosyltransferase gene were integrated at the site. wbgO Or ldhA、wecB、manX、manZ β-1,4-galactosyltransferase gene with 4 copies at site 4 HP3 Strains MG03, K12, and MG1655Δ were obtained respectively. lacZ Δ wcaJ Δ ugd Δ rpH :: galE Δ ybeQ :: galU Δ ldhA :: wbgO Δ wecB :: wbgO Δ manY :: wbgO Δ manZ :: wbgOAnd MG04: K12 MG1655Δ lacZ Δ wcaJ Δ ugd Δ rpH : galE Δ ybeQ :: galU Δ ldhA :: HP3 Δ wecB :: HP3 Δ manX :: HP3 Δ manZ :: HP3 ; (4) Based on MG03, respectively nagB、poxB、ygaY Site integration 3 copy number lgtA , strain MG07 was obtained; (5) Based on MG04, respectively nagB、poxB、ygaY Site integration 3 copy number lgtA , strain MG11 was obtained; (6) In MG07 and MG11 respectively arsB CI-bbhI was integrated at the site, resulting in strains MG08 and MG12, respectively.

[0097] To knock out lacZ For example, the gene knockout methods mentioned above include the following steps: Amplification was performed using wild-type K12 MG1655 as a template. lacZThe upstream homologous arm (500 bp) and downstream homologous arm (500 bp) were tested. The upstream homologous arm PCR system consisted of: 2.5 μL K12 MG1655 template, 2.5 μL each of lacZ-up-F and lacZ-up-R, 25 μL 2×High fidelity Master mix, and 17.5 μL sterile water, for a total of 50 μL. The amplification program was the same as the standard amplification program in Table 5. The downstream homologous arm PCR system consisted of: 2.5 μL K12 MG1655, 2.5 μL each of lacZ-down-F and lacZ-down-R, 25 μL 2×High fidelity Master mix, and 17.5 μL sterile water, for a total of 50 μL. The amplification program was the same as the standard amplification program in Table 5. If the PCR product was free of contaminants, it was purified using a universal DNA purification and recovery kit. If contaminants were present, the product was excised and recovered from the gel or amplified again as needed. The upstream and downstream homologous arms were assembled using fusion PCR. The PCR system was as follows: 2.5 μL upstream homologous arm, 2.5 μL downstream homologous arm, 2.5 μL lacZ-up-F, 2.5 μL lacZ-down-R, 25 μL 2×Highfidelity Master mix, and 15 μL sterile water, for a total of 50 μL. The fusion PCR reaction procedure is shown in Table 5. If the PCR product had no impurities, it was purified using a universal DNA purification and recovery kit; if impurities were present, the product was recovered by gel excision or re-amplified as needed.

[0098] Constructing the pTargetF plasmid: To knock out lacZ For example, using the original pTargetF plasmid as a template, PCR amplification was performed. The PCR system was as follows: 2.5 μL pTargetF plasmid, 2.5 μL each of primers lacZ-N23-F and lacZ-N23-R, 25 μL 2×High Fidelity Mastermix, and 17.5 μL sterile water, for a total of 50 μL. The amplification program was as per Table 5 (standard amplification program). If no contaminants were found, the PCR product was purified using a universal DNA purification and recovery kit (TIANGEN). Then, 1 μL of Dpn I was added and incubated at 37°C for 30 min to digest the template plasmid, followed by another PCR product purification. If contaminants were found, gel extraction was sufficient, or the PCR product was amplified again as needed. The linear plasmid was transformed into DH5α competent cells and circularized. The resulting single clones were activated in liquid, plasmids were extracted, and sequencing was performed for verification.

[0099] 100 ng each of the fused homologous fragment and pTargetF plasmid were electroporated into K12 MG1655+pCpf1 (kana-resistant) competent cells. Immediately after electroporation, 1 mL of antibiotic-free LB medium was added, and the cells were incubated at 37°C and 220 rpm for 1 h. The bacterial culture was then plated on LB+kana (100 mg / L)+spec (100 mg / L) plates and incubated at 37°C and 220 rpm for 12-16 h. Single colonies were picked for colony PCR verification to confirm gene knockout. The colony PCR verification system was as follows: 1 μL of lacZ-YZ-F primer, 1 μL of lacZ-YZ-R primer, 10 μL of 2×Hieff PCR Master mix, and 8 μL of sterile water, for a total of 20 μL. The amplification program is shown in Table 5 (standard amplification program).

[0100] Complete the gene knockout in step (1) using the same method. 。

[0101] In the genome nagB Site integration lgtA For example, the gene integration methods mentioned above include the following steps: by lgtA Using the gene as a template, amplification was performed using primers lgtA-F and lgtA-R to obtain... lgtA ,and nagB The upstream homologous arm (500 bp) and the downstream homologous arm (500 bp) are fused. The fusion method is as follows: Step 1: Pre-fusion: lgtA 2.5 μL, nagB 2.5 μL of upstream homologous arm nagB The downstream homologous arm (2.5 μL), 2×High Fidelity Master mix (25 μL), and sterile water (12.5 μL) were added and subjected to 10 cycles. The second fusion step involved adding 2.5 μL of nagB-up-F and 2.5 μL of nagB-up-R to the above mixture and performing 30 cycles. The fusion PCR reaction program is shown in Table 5. The PCR products were then excised and recovered from the gel.

[0102] Construction of pTargetF plasmid: Using the original pTargetF plasmid as a template, PCR amplification was performed. The PCR system was as follows: 2.5 μL pTargetF plasmid, 2.5 μL each of primers nagB-N23-F and nagB-N23-R, 25 μL 2×High Fidelity Mastermix, and 17.5 μL sterile water, for a total of 50 μL. The amplification program was as per Table 5. If no contaminants were observed, the PCR product was purified using a universal DNA purification and recovery kit (TIANGEN). Then, 1 μL of Dpn I was added and incubated at 37°C for 30 min to digest the template plasmid, followed by another PCR product purification. If contaminants were observed, gel extraction was sufficient, or the PCR product was amplified again as needed. The linear plasmid was transformed into DH5α competent cells and circularized. The resulting single colonies were activated in liquid form, plasmids were extracted, and sequencing was performed for verification.

[0103] 100 ng of the fused gene fragment and pTargetF plasmid were electroporated into K12MG1655 competent cells containing pCpf1 plasmid. The subsequent strain culture and verification process was the same as the gene knockout process.

[0104] Complete the gene integration in steps (2)-(5) in the same way.

[0105] Step (6) CI- bbhI The method for integration into the LNT / LNnT engineered bacteria genome is as follows: CI synthesized by Genewiz bbhI Expression plasmids ( Figure 1 In the figure, CI857-pRpL (i.e., the CI-PRL temperature-controlled promoter) is used as a template. Using primers CI-BbhI-F and CI-BbhI-R, CI- is amplified to obtain CI- BbhI Fragments were amplified using K12 MG1655 as a template, with either arsB-lgtA-up-F / arsB-lgtA-up-R or arsB-lgtA-down-F / arsB-lgtA-down-R as the template. arsB Upstream and downstream homologous arms of the site. Using CI- bbhI Fragments and arsB Using upstream and downstream homologous arms as templates, the homologous fragment CI- was amplified using primers arsB-lgtA-up-F / arsB-lgtA-down-R. bbhI ( arsB The total volume was 50 μL, including CI- bbhI Fragments and arsB2.5 μL each of the upstream and downstream homologous arms, 2.5 μL each of the primers, 25 μL of 2×High Fidelity Master mix, and sterile water to bring the volume to 50 μL. Refer to Table 5 for the standard amplification procedure.

[0106] pTargetF-N23-CI- bbhI ( arsB Plasmid construction: Using pTargetF as a template, and arsB-lgtA-N23-F and arsB-lgtA-N23-R as primers, pTargetF-N23 was amplified. Using pTargetF-N23 as a template, the linear DNA fragment of pTargetF-N23 was amplified using TVF / R primers. The linearized vector pTargetF-N23 and the target fragment CI- were ligated using 2×Seamless Cloning Mix (Biomed). bbhI ( arsB According to Table 6, add each component sequentially to a 0.2 mL PCR tube, mix gently, centrifuge for a few seconds, and incubate at 50℃ for 15 min on a PCR instrument. After the reaction, add 5-10 μL of the ligation product to freshly thawed DH5α competent cells, mix gently, and incubate on ice for 20-30 min. Heat shock at 42℃ for 90 s, then place in an ice-water bath for 2 min, add 1 mL of LB medium, and incubate at 37℃ and 200 rpm for 60 min. After centrifugation, discard part of the supernatant, retaining only 100-200 μL, resuspend the bacterial pellet, and spread the bacterial solution on LB solid culture dishes containing spectinomycin, incubating overnight at 37℃. After single colony activation, sequence to confirm the correct sequence. Extract plasmid pTargetF-N23-CI- bbhI ( arsB ),spare.

[0107] pTargetF-N23-CI- bbhI ( arsB Electroporation was performed on MG07 / MG11 competent cells containing pCpf1 plasmid in step (5), and single colonies were picked and verified with primers arsB-YZ-F / R to obtain strains MG08 and MG12.

[0108] By replacing CI-PRL (temperature-controlled promoter) with promoter J23119, and following the same steps as described above, strains MG08-1 and MG12-1 were obtained.

[0109] The primer information related to the construction process is shown in Table 4, the DNA fragment fusion amplification procedure is shown in Table 5, the vector fragment recombination ligation reaction system is shown in Table 6, and the specific information of the strains is shown in Table 7.

[0110] Table 4 Primers used in the construction of engineered strains

[0111] Table 5 Amplification Procedure

[0112] Table 6. Vector fragment recombination and ligation reaction system

[0113] Note: The typical amount of carrier used is 50-100 ng, and the molar ratio of carrier to fragment is 1:1 to 1:3.

[0114] Table 7 Information on LNT / LNnT engineered strains

[0115] Example 2: Shake-flask fermentation verification Using an inoculation loop, streaked strains MG07, MG08, MG08-1, MG11, MG12, and MG12-1 constructed in Example 1 onto LB agar plates and incubated at 37°C for approximately 15 h. The plate-activated strains were then inoculated into test tubes containing 4 mL of LB medium and incubated overnight at 200 rpm and 37°C to obtain seed culture. 500 μL of seed culture was inoculated into 25 mL of DM medium, with MG08 and MG12 divided into two groups, incubated at 42°C and 37°C for approximately 6 h respectively, both at 200 rpm; the remaining strains were incubated at 200 rpm and 37°C for approximately 6 h. When the cell density (OD) reached... 600 To reach a final concentration of 0.6-0.8, add D-lactose to a final concentration of 8 g / L. Then, transfer the shake flasks to a shaker at 200 rpm and 30°C and incubate for another 96 h.

[0116] LNTri II, LNT, and LNnT in the fermentation broth could all be detected by high-performance liquid chromatography (HPLC). Sample preparation: Take 2 mL of fermentation broth, centrifuge at 12,000 g for 10 min, collect the supernatant, and filter through a 0.22 μm PES aqueous filter membrane. HPLC detection conditions: Detected using an HPLC system (Waters e2695) and Carbohydrate Analysis (Rezex ROA-organic acid H2O)... +(8%) The chromatographic column was used to analyze the sample, with a differential detector. Mobile phase: 0.5 mmol / L H2SO4; flow rate: 0.6 mL / min; column temperature: 60℃; injection volume: 10 μL. Among them, the elution time of LNTri II was 8.2-8.3 min, the elution time of LNT was 7.8-7.9 min, and the elution time of LNnT was 7.6-7.7 min.

[0117] The results of shake-flask fermentation are shown in Table 8.

[0118] Table 8 Results of shake-flask fermentation

[0119] Based on the above shake-flask fermentation results, CI- bbhI After the expression cassette was integrated into the genome, the residual amount of the byproduct LNTri II was significantly reduced during shake-flask fermentation, while the yield of LNT / LNnT increased. Furthermore, the biomass and yield of the strain were slightly lower at an initial culture temperature of 42℃ compared to 37℃; however, an initial culture temperature of 37℃ did not affect the growth of MG08 and MG12 strains, and the temperature-controlled promoter CI857-pRpL could be activated. BbhI The expression of LNT / LNnT is improved, and the yield of LNT / LNnT is increased; MG08-1 and MG12-1 are started using the constitutive promoter J23119. BbhI While expressing LNTri II can reduce the residual amount of the byproduct, LNT / LNnT, it also significantly reduces the yield of LNT / LNnT.

[0120] High levels of residual LNTri II were detected in the fermentation broths of strains MG07 and MG11. This result indicates that in these engineered strains, the supply level of precursor LNTri II exceeds the utilization capacity of the downstream LNT synthesis pathway; therefore, intracellular accumulation of LNTri II is not a bottleneck factor limiting further LNT / LNnT synthesis.

[0121] Example 3 Optimization bbhI Expression temperature In this embodiment, in order to further improve the fermentation yield of strains MG08 and MG12, optimization was performed. BbhI To illustrate the expression, this patent tested different initial culture temperatures. In this embodiment, each strain was divided into 8 fermentation groups, with initial culture temperatures of 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, and 42℃, respectively. The fermentation process and detection methods are as follows: Primary seed culture: Drip bacterial culture from the glycerol preservation tube and streak it onto an LB solid culture dish. Incubate at 37°C for 10-12 h. Use an inoculation loop to scrape one loopful and inoculate it into 4 mL of LB liquid medium. Incubate at 37°C for 200 rpm for 10-12 h to obtain the primary seed culture.

[0122] Secondary seed culture: Transfer the above primary seed culture medium at an inoculation rate of 10% (v / v) to a 500 mL shake flask containing 40 mL of DM medium, and incubate at 37℃ and 200 rpm for 3-5 h until OD reaches the target value. 600 =1.2-1.5, to obtain secondary seed culture medium.

[0123] The secondary seed culture was inoculated at 2% (v / v) into a 5 L fermenter containing an initial volume of 2 L of DM medium and cultured at 35-42℃. Initial culture conditions: pH: 6.80±0.05 (adjusted with ammonia); agitator: 50 rpm; aeration rate: 1 vvm. Cultured until OD... 600 =20-25℃, cool down to 30℃ (to control the temperature-controlled promoter to terminate BbhI expression), add D-lactose in batches, and control the residual amount <2 g / L. During fermentation, control dissolved oxygen at 25±5%. After the glycerol in the culture medium is almost completely consumed, add glycerol in batches, and control the residual amount <1 g / L. End fermentation at approximately 75 h.

[0124] HPLC Detection: Glycerol, lactose, LNTri II, LNT, and LNnT in the fermentation broth can all be detected by high-performance liquid chromatography (HPLC). Sample Preparation: Take 2 mL of fermentation broth, centrifuge at 12,000 g for 10 min, collect the supernatant, and filter through a 0.22 μm PES aqueous filter membrane. HPLC Detection Conditions: HPLC was performed using a Waters e2695 system and a Carbohydrate Analysis (Rezex ROA-organic acid H2O) system. + (8%) The chromatographic column was used to analyze the sample, with a differential detector. Mobile phase: 0.5 mmol / L H2SO4; flow rate: 0.6 mL / min; column temperature: 60 ℃; injection volume: 10 μL. The peak times for LNTri II were 8.2-8.3 min, LNT was 7.8-7.9 min, and LNnT was 7.6-7.7 min.

[0125] The fermentation results are shown in Tables 9 and 10.

[0126] Table 9. Fermentation results of MG08 at different initial culture temperatures.

[0127] Table 10 Fermentation results of MG12 at different initial culture temperatures

[0128] This embodiment aims to screen a suitable temperature range that does not affect strain growth or the leakage-induced expression of BbhI by the temperature-controlled promoter. Fermentation results at different initial culture temperatures are shown in Tables 9 and 10. At initial culture temperatures of 35°C and 36°C, BbhI expression was low, and the degradation of the byproduct LNTri II was incomplete. At initial culture temperatures of 37°C-42°C, BbhI expression was sufficient to completely degrade the byproduct LNTri II, but the yields of LNT and LNnT decreased with increasing temperature. This may be because excessively high BbhI expression caused the metabolic flux of LNTri II to be biased towards degradation into lactose rather than the generation of LNT and LNnT. In summary, the optimal initial culture temperature of 37°C resulted in the highest yields of LNT (94.17 g / L) and LNnT (35.18 g / L) with no LNTri II accumulation, which is beneficial for the purification and production of the target product.

[0129] Example 4 Optimization bbhI expression time In this embodiment, to further improve the fermentation yield of strains M08 and MG12, the OD of the strains during cooling was optimized. 600 This led to optimization bbhI The expression intensity was determined. In this example, four fermentation groups were set up for each strain, with two replicates per group. The preparation process of the culture medium and seed culture was the same as in Example 3. The secondary seed culture was inoculated at 2% (v / v) into a 5 L fermenter with an initial volume of 2 L of DM medium and cultured at 37°C. When OD... 600 When the temperature falls within one of the four ranges: <15, 15-25, 25-40, and >40, the culture temperature should be lowered to 30°C to terminate the incubation. bbhI Expression. Other fermentation process controls were consistent with Example 3, and the detection methods were also consistent with Example 3. Different OD values... 600 The yield and by-product residues after cooling are shown in Tables 11 and 12.

[0130] Table 11 Different OD 600 Fermentation data of MG08 during cooling

[0131] Table 12 Different OD 600 Fermentation data of MG12 during cooling

[0132] As shown in Tables 11 and 12, the OD during cooling... 600 When the yield is less than 15%, although the yield is high, about 10% of byproducts remain, which is not conducive to subsequent purification; OD600 The highest yield was achieved between 15 and 25 degrees Celsius, with no byproduct residue; OD 600 At 25-40°C, the yield is high and there are no by-product residues; OD 600 When the temperature exceeds 40°C, the yield decreases significantly. In summary, the optimal cooling OD... 600 It is 15-25.

[0133] Comparative Example 1: Based on plasmid expression bbhI dual-strain fermentation system CI- bbhI Using BbhI-F / R as the template, the primers amplify the fragment. bbhI Using pETDuet-1 as a template and pETDuet-F / R as primers, the linear vector pETDuet-1 was amplified. The linearized vector pETDuet-1 and the target fragment were ligated using 2×Seamless Cloning Mix (Biomed). bbhI plasmid pETDuet-1- was obtained. bbhI The primer sequences are shown in Table 13.

[0134] Using Escherichia coli BL21(DE3) as a chassis, plasmid pETDuet-1- was introduced. bbhI The strain BL21(DE3), pETDuet-1- bbhI The seed culture was obtained by inoculating 100 mL of LB medium containing ampicillin and incubating overnight at 37°C. The seed culture was then inoculated at a ratio of 1% into DM medium and cultured in shake flasks at 37°C and 200 rpm until OD (dose elapsed). 600 When the concentration is around 0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM for induction, and continue culturing at 25℃ for 8-10 h to obtain pre-cultured BL21(DE3), pETDuet-1-bbhI bacterial suspension.

[0135] The LNT / LNnT production strain (MG07 or MG11) was cultured at 37°C until the strain reached OD2000 under the optimized fermentation conditions described in Examples 3-4. 600 When the temperature drops to 30°C at 15-25°C, the culture continues. All other control conditions are the same as in Example 3 or Example 4. After fermentation for 72 hours, 20% (volume ratio) of the pre-cultured BL21(DE3), pETDuet-1-bbhI bacterial solution is added to the fermentation broth of MG07 or MG11 production strain, and the mixed strain is cultured for another 3-5 hours.

[0136] The detection methods for glycerol, lactose, LNTriII, LNT, and LNnT in the fermentation products were the same as in Example 3, and the results are as follows: Figure 2-3 As shown.

[0137] Table 13

[0138] Results of batch-fed dual-strain fermentation in a 5 L fermenter of MG07 are shown below. Figure 2 The highest yield of LNT was 81 g / L, lower than the fermentation yield of MG08 (111.65 g / L); the results of fed-batch dual-strain fermentation of MG11 in a 5 L fermenter are shown in [reference needed]. Figure 3 The highest yield of LNnT was 29 g / L, lower than the fermentation yield of MG12 (38.99 g / L). The addition of BL21 (DE3) and pETDuet-1... bbhI After fermentation, LNTriII was rapidly degraded, and no residual LNTriII was detected in the final mixed culture, but the yields of LNT and LNnT were reduced. Furthermore, this fermentation process is relatively complex to implement, requiring strict control of strain BL21(DE3), pETDuet-1- bbhI The inoculation and transplanting times, the dual-strain fermentation increased the raw material cost, and the use of the antibiotic ampicillin during the fermentation process affected product safety.

[0139] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. An engineered bacterium producing lactose-N-tetrasaccharide and / or lactose-N-neotetrasaccharide, characterized in that, The engineered bacteria express a temperature-controlled promoter and β-acetylhexosaminease.

2. The engineered bacteria according to claim 1, characterized in that, The temperature-controlled promoter includes CI857-pRpL; preferably, the temperature-controlled promoter includes the sequence shown in SEQ ID No. 12 or a nucleotide sequence having at least 95% identity with SEQ ID No.

12.

3. The engineered bacteria according to claim 1, characterized in that, The β-acetylhexosamine enzyme comprises the sequence shown in SEQ ID No. 14 or an amino acid sequence having at least 95% identity with SEQ ID No.

14.

4. The engineered bacteria according to any one of claims 1-3, characterized in that, The engineered bacteria are selected from one or more of Escherichia coli, Streptococcus, Bacillus, Saccharomyces cerevisiae, and Pichia pastoris.

5. The engineered bacteria according to any one of claims 1-3, characterized in that, The engineered bacteria genome integrates and expresses a temperature-controlled promoter and β-acetylhexosaminease.

6. A biomaterial, characterized in that, The biomaterial includes any one of the following B1)-B3): B1) Expression cassette, which expresses a temperature-controlled promoter and β-acetylhexosaminease; B2) A recombinant vector containing the expression cassette described in B1); B3) Whole-cell catalyst, wherein the whole-cell catalyst contains the expression cassette of B1), the recombinant vector of B2), and / or the engineered bacteria as described in any one of claims 1-5.

7. A method for preparing lactose-N-tetrasaccharide and / or lactose-N-neotetrasaccharide, characterized in that, The method includes: culturing the engineered bacteria as described in any one of claims 1-5.

8. The method according to claim 7, characterized in that, The culture temperature is 37℃-42℃.

9. The method according to claim 7 or 8, characterized in that, The cultured OD 600 The expression of β-acetylhexosamine enzyme is stopped when the value is 15-40; preferably, the cessation of expression of β-acetylhexosamine enzyme is achieved by lowering the culture temperature; more preferably, the culture temperature is lowered to ≤30℃.

10. The use of the engineered bacteria as described in any one of claims 1-5 or the biological material as described in claim 6 in any one or more of the following C1)-C5): C1) Production of lactose-N-tetrasaccharide and / or lactose-N-neotetrasaccharide; C2) Increase the yield of lactose-N-tetrasaccharide and / or lactose-N-neotetrasaccharide; C3) Improve the purity of lactose-N-tetrasaccharide and / or lactose-N-neotetrasaccharide; C4) Reduces the content of the byproduct lactose-N-trisaccharide; C5) Increase the yield of lactose-N-tetrasaccharide and / or lactose-N-neotetrasaccharide while eliminating lactose-N-trisaccharide.

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