Application of transporter in improving yield and / or purity of lactose-N-neotetraose

Abstract

The invention provides an application of a transporter in improving the yield and / or purity of lactose-N-neotetraose (LNnT), and relates to the technical field of genetic engineering. Transporter proteins of different sources are screened, the transporter protein which aims at LNnT synthesis and can achieve efficient transportation is obtained, and a new biological material is provided for LNnT synthesis. Moreover, the efficient transporter obtained by screening is used for transporting the LNnT to the outside of cells, so that the pressure on the cells caused by overhigh concentration of intracellular products is relieved, the conversion of an intermediate product LNT II to the LNnT is improved to the maximum extent, the yield is improved while byproducts are reduced, the yield of a byproduct lactose-N-neohexaose is also reduced, and the separation and purification of later products are facilitated.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering technology, and in particular to the application of transport proteins in improving the yield and / or purity of lactose-N-neotetraose. Background Art

[0002] Breast milk contains a variety of active biomolecules, such as oligosaccharides, immunoglobulins, hormones and growth factors, which provide comprehensive and important nutrients for the growth and development of young children. Human milk oligosaccharides (HMOs) are a general term for complex oligosaccharides with unique and diverse structures in breast milk, which have rich physiological functions. To date, more than 200 different types of HMOs have been identified, which are divided into three categories: neutral fucosylation, neutral non-fucosylation and acidic sialylated sugars. Among the neutral fucosylated oligosaccharides, lactose-N-neotetraose (LNnT) and lactose-N-tetraose (LNT) are the core components of HMOs, accounting for about 10% of the total human milk oligosaccharides, and are also the hot spots of current research.

[0003] LNnT and LNT are oligoisomers composed of monomers D-glucose, D-galactose and N-acetylglucosamine. They can also be used as structural units for derivatization. By connecting L-fucose to LNnT and / or LNT with different glycosidic bonds, a series of complex fucosylated and / or sialylated human milk oligosaccharides are formed, accounting for about 20% of the total human milk oligosaccharides.

[0004] Among the various LNnT and LNT synthesis methods, microbial synthesis based on metabolic engineering is the most promising method. Using microorganisms as host strains, it can use lactose to produce high-value compounds, demonstrating the attributes of economic feasibility, efficiency, and environmental sustainability. Both synthetic LNnT and LNT are synthesized by two-step reactions, and lactose-N-triose II (Lacto-N-triose II, LNT II) is the common precursor of both and the main by-product in the fermentation process. When LNT II is linked to the acceptor substrate with a β-1,4 bond, it can be converted to LNnT; when it is linked to the acceptor substrate with a β-1,3 bond, it can be converted to LNT.

[0005] In addition, due to the mixed substrates of glycosyltransferase, a long and thin sugar chain structure is formed. The researchers speculate that LNnT can also serve as a substrate for β-1,3-N-acetylglucosamine transferase to form an intermediate pentasaccharide, which is further converted into pLNnH (lactose-N-neohexose) by β-1,4-galactosyltransferase. Similarly, pLNnH can serve as a substrate for the next cycle reaction, eventually forming pLNnO (lactose-N-neooctaose), thus affecting the quality of the final product.

[0006] As early as 2002, researchers used Escherichia coli JM109 as the chassis cell to construct a synthetic strain of LNnT, accompanied by the production of byproduct LNT II, ​​and found poly-N-acetyllactosamine in the fermentation product. In subsequent studies, Escherichia coli BL21 star (DE3) was developed through system metabolic engineering to synthesize LNnT. After strain screening, efficient β-1,4-GalT identification, key gene expression regulation and enhanced UDP-galactose supply, 22.07g / L LNnT and 19.78g / L LNT II were produced. In addition to causing low purity of the final product and increasing separation and purification costs, the high proportion of LNT II will also result in low conversion rate of the substrate lactose and increased raw material costs. It has been reported that the production of LNT II increased significantly after overexpression of endogenous E. coli transport genes setA, setB, ydeA and mdfA. At the same time, some researchers have found that knocking out any one or more of the setA, setB, ydeA and mdfA genes can increase the production of LNnT and reduce the byproduct LNT II. However, some studies believe that when LNT II is exported from E. coli to the extracellular environment, it cannot be internalized into the cell and effectively converted into LNnT. Improving the conversion rate of LNT II and enhancing carbon source utilization are crucial to the entire LNnT production process. Therefore, the selection of transport proteins is crucial to reducing the content of byproducts in the LNnT production process, and it is urgent to discover more transport proteins with excellent functions to improve the purity and production of LNnT products. Summary of the invention

[0007] With reference to previous studies, the present invention aims to solve the problem of excessive by-products in LNnT synthetic strains, comprehensively considers the effects of transporter knockout and integration on strain product synthesis, screens LNnT transporters from different sources for integration, transports LNnT synthesized intracellularly to the extracellular space, reduces cell pressure while enhancing LNT II utilization, timely excretes LNnT to reduce the generation of long-chain structures, increases LNnT production, reduces by-products, and improves LNnT cell stability.

[0008] On the one hand, the present application provides the use of a transporter in improving the yield and / or purity of lactose-N-neotetraose, wherein the transporter comprises at least any one or more of the following A1)-A8):

[0009] A1) a transporter derived from Serratia fonticola; preferably, the transporter derived from Serratia fonticola is SfSET; more preferably, the SfSET comprises an amino acid sequence as shown in SEQ ID NO.10 or an amino acid sequence having 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%, 99.9% identity with SEQ ID NO.10;

[0010] A2) a transporter derived from Escherichia fergusonii; preferably, the transporter derived from Escherichia fergusonii is EMFST; more preferably, the EMFST comprises an amino acid sequence as shown in SEQ ID NO.12 or an amino acid sequence having 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%, 99.9% identity with SEQ ID NO.12;

[0011] A3) a transporter derived from Chimaeribacter californicus; preferably, the transporter derived from Chimaeribacter californicus is CaMFST; more preferably, the CaMFST comprises an amino acid sequence as shown in SEQ ID NO.14 or an amino acid sequence having 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%, 99.9% identity with SEQ ID NO.14;

[0012] A4) a transporter derived from unclassified Serratia (MFS transporter); preferably, the transporter derived from unclassified Serratia (MFS transporter) is uSSET; more preferably, the uSSET comprises an amino acid sequence as shown in SEQ ID NO.16 or an amino acid sequence having 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%, 99.9% identity with SEQ ID NO.16;

[0013] A5) a transporter derived from Gibbsiella quercinecans; preferably, the transporter derived from Gibbsiella quercinecans is GqSET; more preferably, the GqSET comprises an amino acid sequence as shown in SEQ ID NO.18 or an amino acid sequence having 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%, 99.9% identity with SEQ ID NO.18;

[0014] A6) a transporter derived from Kosakonia pseudosacchari; preferably, the transporter derived from Kosakonia pseudosacchari is KpSET; more preferably, the KpSET comprises an amino acid sequence as shown in SEQ ID NO.20 or an amino acid sequence having 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%, 99.9% identity with SEQ ID NO.20;

[0015] A7) a transporter derived from unclassified Serratia (sugar efflux transporter); preferably, the transporter derived from unclassified Serratia (sugar efflux transporter) is uSMFST; more preferably, the uSMFST comprises an amino acid sequence as shown in SEQ ID NO.22 or an amino acid sequence having 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%, 99.9% identity with SEQ ID NO.22;

[0016] A8) a transporter derived from Serratia oryzae; preferably, the transporter derived from Serratia oryzae is SoMFST; more preferably, the SoMFST comprises the amino acid sequence as shown in SEQ ID NO.24 or an amino acid sequence having 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%, 99.9% identity with SEQ ID NO.24.

[0017] The lactose-N-neotetraose is extracellular lactose-N-neotetraose.

[0018] On the other hand, the present application also provides a biomaterial, which comprises at least one of the following B1)-B3):

[0019] B1) a nucleic acid molecule encoding the transporter;

[0020] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0021] B3) A recombinant vector containing the nucleic acid molecule described in B1) and / or a recombinant vector containing the expression cassette described in B2).

[0022] Furthermore, the nucleic acid molecule described in B1) includes any one or more of C1)-C9):

[0023] C1) the nucleotide sequence comprises the nucleic acid molecule shown in SEQ ID NO.9;

[0024] C2) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO.11;

[0025] C3) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO.13;

[0026] C4) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO.15;

[0027] C5) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO.17;

[0028] C6) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO.19;

[0029] C7) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO.21;

[0030] C8) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO.23;

[0031] C9) A nucleic acid molecule having 80% or more identity with the nucleic acid molecule of any one of C1) to C8).

[0032] In a specific embodiment,

[0033] The nucleic acid molecule encoding the SfSET comprises the nucleotide sequence as shown in SEQ ID NO.9 or a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 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%, 99.9% identity with SEQ ID NO.9;

[0034] The nucleic acid molecule encoding the EMFST comprises the nucleotide sequence as shown in SEQ ID NO.11 or a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 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%, 99.9% identity with SEQ ID NO.11;

[0035] The nucleic acid molecule encoding the CaMFST comprises the nucleotide sequence as shown in SEQ ID NO.13 or a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 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%, 99.9% identity with SEQ ID NO.13;

[0036] The nucleic acid molecule encoding the uSSET comprises the nucleotide sequence as shown in SEQ ID NO.15 or a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 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%, 99.9% identity with SEQ ID NO.15;

[0037] The nucleic acid molecule encoding the GqSET comprises the nucleotide sequence as shown in SEQ ID NO.17 or a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 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%, 99.9% identity with SEQ ID NO.17;

[0038] The nucleic acid molecule encoding the KpSET comprises the nucleotide sequence as shown in SEQ ID NO.19 or a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 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%, 99.9% identity with SEQ ID NO.19;

[0039] The nucleic acid molecule encoding the uSMFST comprises the nucleotide sequence shown in SEQ ID NO.21 or a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 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%, 99.9% identity with SEQ ID NO.21;

[0040] The nucleic acid molecule encoding the SoMFST comprises the nucleotide sequence as shown in SEQ ID NO.23 or a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 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%, 99.9% identity to SEQ ID NO.23.

[0041] In this application, 8 new transporters were screened for the LNnT production process. The expression of the selected transporters in this process can effectively transport LNnT synthesized in the cell to the outside of the cell, reduce cell pressure, that is, it can improve the utilization of the intermediate product LNT II and timely expel LNnT to reduce the synthesis of long-chain structure byproducts and improve LNnT cell stability. Furthermore, the above-mentioned transporters can be used in the LNnT production process to effectively increase the yield of lactose-N-neotetraose and the purity of the product.

[0042] Those skilled in the art will appreciate that other commonly used expression elements can be adaptively added to the expression cassette described in the present application to assist in the expression of the target gene, such as adding tags, fluorescent protein markers, resistance screening markers, etc.

[0043] A person skilled in the art can select a known plasmid to construct a recombinant vector according to actual conditions and perform conventional optimization and modification on the constructed recombinant vector. The present application does not impose mandatory restrictions on the type of plasmid. In a preferred embodiment, the vector can be a pEcCpf1 plasmid, a pcrEG plasmid, or the like.

[0044] In an optional embodiment, the expression cassette of the target gene is located on a recombinant vector or is introduced into a recombinant microorganism or a recombinant cell using a recombinant vector.

[0045] On the other hand, the present application also provides a recombinant microorganism that can secrete and express lactose-N-neotetraose, wherein the recombinant microorganism comprises any one or more of the following D1)-D4):

[0046] D1) a recombinant microorganism containing the transporter protein;

[0047] D2) a recombinant microorganism containing the nucleic acid molecule described in B1);

[0048] D3) a recombinant microorganism containing the expression cassette described in B2);

[0049] D4) A recombinant microorganism containing the recombinant vector described in B3).

[0050] Furthermore, the recombinant microorganism is any one of the following E1)-E2):

[0051] E1) Escherichia bacteria;

[0052] E2) Escherichia coli.

[0053] Preferably, the microorganism is Escherichia coli; more preferably, the microorganism is Escherichia coli (E. coli) MG1655.

[0054] More preferably, the recombinant microorganism further comprises one or more of the following F1)-F3) modifications:

[0055] F1) silencing or reducing the expression of lacZ, wcaJ and / or ugd; preferably, knocking out the lacZ, wcaJ and / or ugd genes;

[0056] F2) expressing or overexpressing galE, lgtA and / or galT3;

[0057] F3) expressing or overexpressing the transporter.

[0058] Preferably, the galE gene is integrated at the wcaC site; more preferably, the galE is galE derived from Escherichiacoli, the amino acid sequence of the galE is shown in SEQ ID NO.2, and the nucleotide sequence encoding galE is shown in SEQ ID NO.1.

[0059] Preferably, the copy number of the lgtA gene is 1-3; more preferably, the lgtA gene is integrated at the arsB, nagB and / or poxB sites; more preferably, the lgtA is lgtA derived from Neisseria spp., the amino acid sequence of the lgtA is shown in SEQ ID NO.4, and the nucleotide sequence encoding lgtA is shown in SEQ ID NO.3;

[0060] Preferably, the copy number of galT3 is 1-4; more preferably, the galT3 gene is integrated at the ldhA, manX, manZ and / or wecB sites; preferably, the galT3 is galT3 derived from Helicobacter spp., the amino acid sequence of the galT3 is shown in SEQ ID NO.6, and the nucleotide sequence encoding galT3 is shown in SEQ ID NO.5.

[0061] Preferably, the integration site of the nucleic acid molecule encoding the transporter is hlyE.

[0062] It is understandable to those skilled in the art that the above transformation can be accomplished using existing gene editing techniques. In a preferred embodiment, the above transformation is accomplished using CRISPR-Cpf1-assisted homologous recombination methods.

[0063] On the other hand, the present application also provides a whole-cell catalyst for preparing lactose-N-neotetraose, wherein the whole-cell catalyst comprises the recombinant microorganism.

[0064] On the other hand, the present application also provides a method for producing lactose-N-neotetraose, which comprises fermenting and producing lactose-N-neotetraose using the recombinant microorganism or the whole-cell catalyst.

[0065] It will be appreciated by those skilled in the art that a general method can be used to activate and ferment the recombinant microorganism to obtain lactose-N-neotetraose.

[0066] In a preferred embodiment, the method comprises:

[0067] Take the recombinant microorganism and inoculate the seed culture medium, culture at 25℃-40℃, 100-300rpm, culture for 12-24h, inoculate 1%-5% inoculum into the fermentation medium, culture at 25℃-40℃, 100-300rpm, OD 600 When it reaches 0.6-0.8, add 1-5g / L lactose, lower the culture temperature to 20℃-30℃, and continue to culture for 36-72h.

[0068] In a preferred embodiment, the method comprises:

[0069] Take the recombinant microorganism and inoculate it into the seed culture medium, culture it at 25℃-40℃, 100-300rpm, and culture it for 12-24h. Inoculate it into the seed culture medium at a rate of 1%-5%. 600 When the growth reaches 1.5-2.0, it is inoculated into a fermentation tank containing fermentation medium for fermentation. When inoculation, 1-5g / L sterile thiamine is added, and the addition amount is 1-5mL / L. The fermentation conditions are: culture at 25℃-40℃, adjust the pH to 6.6-6.8, ventilation volume 0.1-0.5vvm, and the dissolved oxygen-related rotation speed and ventilation volume are maintained at about 25±5%. After the dissolved oxygen rebounds, glycerol is added to maintain growth. When OD 600 When the concentration reaches 30-40, add 1-5g / L lactose. When the lactose concentration is less than 0.5g / L, start the lactose addition. The initial flow rate should be consistent with the lactose consumption rate. When the lactose accumulation is greater than 1g / L, stop adding lactose immediately and wait until the next point measurement shows that the lactose concentration is less than 0.5g / L before starting again.

[0070] Preferably, the culture medium is an antibiotic-free culture medium.

[0071] Preferably, the seed culture medium is LB culture medium, and the fermentation culture medium is DM culture medium.

[0072] On the other hand, the present application also provides the use of the biological material, the recombinant microorganism, the whole-cell catalyst or the method in increasing the yield of lactose-N-neotetraose.

[0073] The present application demonstrates that the transporters are all capable of increasing the yield of lactose-N-neotetraose, and the yield of lactose-N-neotetraose can reach 19.52 g / L-31.96 g / L, which is greater than the case of only expressing the endogenous transporter (12.18 g / L).

[0074] On the other hand, the present application also provides the use of the biological material, the recombinant microorganism, the whole cell catalyst or the method in reducing by-products in lactose-N-neotetraose products.

[0075] Preferably, the by-products include lactose-N-triose II (LNT II) and lactose-N-neohexose (pLNnH) (ie, neohexaose, lactose-N-neohexose).

[0076] On the other hand, the present application also provides the use of the biological material, the recombinant microorganism, the whole cell catalyst or the method in reducing lactose-N-triose and / or lactose-N-neohexaose in lactose-N-neotetraose products.

[0077] The present invention has the following beneficial effects:

[0078] The invention combines and transforms wild-type Escherichia coli MG1655, knocks out LNnT anabolism competition genes, integrates multiple copies of β-1,3-N-acetylglucosamine transferase and β-1,4-galactosyl transferase, and improves the yield of the product LNnT.

[0079] The present invention screens transport proteins from different sources, obtains transport proteins that can achieve efficient transport for LNnT synthesis, and provides new biomaterials for LNnT synthesis. In addition, the high-efficiency transport proteins obtained by screening are used to transport LNnT to the extracellular space, thereby alleviating the pressure on cells caused by excessively high intracellular product concentrations, maximizing the conversion of the intermediate product LNT II to LNnT, reducing byproducts while increasing yield, and also reducing the yield of byproduct lactose-N-neohexose, thereby facilitating the separation and purification of subsequent products.

[0080] The invention also provides a new LNnT synthetic engineering bacterium, which provides a theoretical basis and new materials for the industrial synthesis of LNnT. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0082] Figure 1 This is a diagram of the 5L fed-batch fermentation process of the KB-027 strain;

[0083] Figure 2 This is the result of 5L fed-batch fermentation of KB-029-KB-037 strain. DETAILED DESCRIPTION

[0084] Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules in molecular evolution research.

[0085] Recombination: Broadly speaking, any gene exchange process that causes genotype changes is called recombination.

[0086] Whole-cell catalyst: 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 corresponding complete biological organism involved in the catalytic process is the whole-cell catalyst.

[0087] Engineered bacteria: Fungal cell strains that use genetic engineering methods to efficiently express exogenous genes.

[0088] (Gene) knockout: It is a foreign DNA introduction technology that uses a DNA fragment containing a certain known sequence to undergo homologous recombination with a gene with the same or similar sequence in the recipient cell genome, integrate into the recipient cell genome and express it. It changes the genetic genes of an organism for a sequence with a known sequence but unknown function, making a specific gene function ineffective, thereby shielding some functions.

[0089] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0090] If not otherwise specified, in the following embodiments, reagents or instruments used without indicating the manufacturer are conventional products that can be purchased commercially. If specific conditions are not indicated in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The plasmids, endonucleases, PCR enzymes, column DNA extraction kits and DNA gel recovery kits used in the following examples are commercial products, and specific operations are carried out according to the kit instructions.

[0091] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields, and can be specifically carried out according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0092] in,

[0093] The Gene ID number of the lacZ gene is 945006, the Gene ID number of the wcaJ gene is 946583, the Gene ID number of the ugd gene is 946571, the Gene ID number of the wcaC locus is 946579, the Gene ID number of the arsB locus is 948011, the Gene ID number of the nagB locus is 945290, the Gene ID number of the poxB locus is 946132, the Gene ID number of the ldhA locus is 946315, the Gene ID number of the manX locus is 946334, the Gene ID number of the manZ locus is 946342, the Gene ID number of the wecB locus is 944789, and the Gene ID number of the hlyE locus is 945745.

[0094] Fermentation (DM) medium (Defined medium, DM) formula: glycerol 30g / L, yeast powder 5g / L, potassium dihydrogen phosphate 13.5g / L, diammonium hydrogen phosphate 4.0g / L, magnesium sulfate heptahydrate 1.4g / L, citric acid monohydrate 1.7g / L, trace element solution 10.0mL / L (ferrous sulfate heptahydrate 10.0g / L, anhydrous calcium chloride 2.0g / L, zinc sulfate heptahydrate 2.2g / L, manganese sulfate monohydrate 0.38g / L, copper sulfate pentahydrate 1.0g / L, dissolved in water, add 5mL concentrated hydrochloric acid to promote dissolution), pH adjusted to 6.8 before sterilization.

[0095] All culture media need to be sterilized at 115°C for 30 min. After sterilization, sterile thiamine (filter sterilized) is added to the culture media at a final concentration of 4.5 mg / L.

[0096] Detection conditions for LNnT, LNT II, ​​neohexasaccharide, lactose and glycerol: The samples were analyzed by high performance liquid chromatography (HPLC) system and Carbohydrate Analysis (AMINEX HPX-87H Organic Acid Analysis Column) column under differential detector. The mobile phase used was 5mmol / LH 2 SO 4 ; Flow rate: 0.6mL / min; Column temperature: 60℃; Injection volume: 10μL.

[0097] Example 1 Construction of LNnT production strain

[0098] In this embodiment, the inventors used the CRISPR-Cpf1 system-assisted homologous recombination method introduced in the paper "Combining CRISPR-Cpf1 and Recombineering Facilitates Fast and Efficient Genome Editing in Escherichia coli" published by Zhu X et al. in 2022 to knock out and integrate the target gene based on Escherichia coli MG1655.

[0099] Using E.coli MG1655 as the chassis cell, knocking out lacZ, wcaJ and ugd genes, and integrating the galE gene (derived from Escherichia coli, the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2) at the wcaC site, the initial chassis KB-015 was obtained. Based on KB-015, the lgtA gene was first integrated into the arsB, nagB, and poxB sites to construct a strain KB-020 with 3 copies of lgtA (derived from Neisseria spp., the nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4). The construction process is the same as the engineering strain construction method disclosed in the Chinese invention patent application document with publication number CN118028129A.

[0100] Using KB-020 as the starting strain, the β-1,4-galactosyltransferase galT3 gene (derived from Helicobacter spp., with a nucleotide sequence as shown in SEQ ID NO.5 and an amino acid sequence as shown in SEQ ID NO.6) was integrated into the ldhA, manX, manZ, and wecB sites to obtain the LNnT-producing engineered strain KB-027.

[0101] The strain construction method involved in the above is:

[0102] (1) The pEcCpf1 plasmid was introduced into the engineered strain KB-020 by heat shock transformation, and a single colony was picked from the plate and inoculated into LB medium containing kanamycin resistance and cultured for 12 h. The cells were transferred to the same medium and induced with arabinose to prepare electrocompetent cells to induce the expression of the λ-Red recombinase system.

[0103] (2) The pcrEG plasmid was amplified by PCR using primers ldhA-N23-F / R, and the resulting linear DNA was circularized into a pcrEG-ldhA plasmid targeting the ldhA site through single-fragment one-step cloning.

[0104] (3) Using the genome of Escherichia coli MG1655 as a template, primers ldhA-up-F / R and ldhA-down-F / R were used to amplify the upstream and downstream fragments of the ldhA site, respectively. Using the galT3 gene as a template, primers J23119-galT3-F / R were used to amplify the galT3 gene fragment by PCR, and gel recovery and purification were performed. Then, the upstream and downstream fragments were connected to the target gene fragment by overlap extension PCR, and finally the donor DNA fragment integrated into the genome was obtained.

[0105] (4) The pcrEG-ldhA plasmid and the donor DNA fragment were transferred into the prepared KB-020 competent cells containing the pEcCpf1 plasmid by electroporation. After electroporation, LB medium was added for recovery culture, and all cells were spread on a double-resistance plate containing kanamycin and spectinomycin, and inverted and cultured at 37°C for 12-15 hours.

[0106] (5) Single colony screening by colony PCR. Pick a number of single colonies, perform internal PCR verification with primers YZ-ldhA-F and YZ-galT3-in-R, and then perform nucleic acid electrophoresis detection. For colonies with bands at the target position, perform external PCR verification with primers YZ-ldhA-F / R. The PCR products of colonies that pass both internal and external verifications are sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. If the sequencing results match the theoretical sequence, the colony has successfully integrated the galT3 gene at the ldhA site.

[0107] (6) The successfully verified strain was inoculated into a test tube containing LB medium containing kanamycin and rhamnose at a final concentration of 10 mM, and cultured for 12 h to remove the pcrEG-ldhA plasmid. Then, the target strain was inoculated into liquid LB medium containing 5 g / L glucose, and the culture solution was streaked on a plate containing 5 g / L glucose and 15 g / L sucrose to remove the pEcCpf1 plasmid. Finally, the full genome strain KB-021 with the galT3 gene integrated at the ldhA site was obtained.

[0108] In addition, the integration of galT3 at the manZ, manX and wecB sites was consistent with the above steps (1)-(6), and finally the engineered bacteria KB-027 with 4 copies of galT3 integrated into the genome was obtained. The specific primers involved are shown in Table 1.

[0109] Table 1 Primers used in key pathway integration examples

[0110]

[0111]

[0112] The shake flask fermentation culture method is:

[0113] A single colony was picked from the plate and inoculated into 4 mL of antibiotic-free LB medium, cultured at 37°C, 200 rpm overnight, the bacterial concentration was determined by a spectrophotometer, and the same inoculum was inoculated into 25 mL of fermentation medium, cultured at 37°C, 200 rpm, and the OD 600 When the pH reaches 0.6-0.8, 5 g / L lactose is added, the culture temperature is lowered to 30°C, and the culture is continued for 72 hours. After the fermentation is completed, 2 mL of the fermentation liquid is taken, centrifuged at 12000 rpm for 10 minutes, and the supernatant is filtered through a 0.22 μm PES water filter membrane to obtain the sample for extracellular detection. The extracellular concentrations of LNnT and LNT II in the sample for extracellular detection are determined by HPLC.

[0114] After testing, the strain KB-020 had the highest yield of byproduct LNT II of 5.18 g / L, the strain KB-027 had a yield of LNnT of 3.47 g / L, and the byproduct LNT II of 0.08 g / L.

[0115] Example 2 Verification of 5L fed-batch fermentation of LNnT genetically engineered bacteria

[0116] In this example, the KB-027 engineered bacteria constructed in Example 1 were used to carry out a 5 L fed-batch fermentation experiment.

[0117] The specific method is as follows: pick a single KB-027 colony from the plate and inoculate it into 4 mL of antibiotic-free LB medium, culture it at 37°C, 200 rpm overnight, inoculate it into 25 mL of LB medium at a 1% inoculum, and wait until the OD 600 When the cells grow to 1.5-2.0, they are inoculated into a 5L fermentation tank containing 2L fermentation medium. When inoculating, 4.5g / L sterile thiamine (VB1) is first added, and the addition amount is 1mL / L.

[0118] The feed solution formula for batch fed fermentation includes feed 1: 600 g / L glycerol, 20 g / L magnesium sulfate heptahydrate, 0.2 g / L thiamine; feed 2: 200 g / L lactose; pH control: 50% ammonia water (v / v).

[0119] The control method is: culture at 30℃, add 50% ammonia water (v / v) through a constant flow pump to maintain the pH of the medium in the tank at 6.6-6.8. The ventilation volume is 0.5vvm, the dissolved oxygen correlation speed and ventilation volume are maintained at about 25±5%, and glycerol (i.e. feed 1) is added after the dissolved oxygen rebounds to maintain growth. When OD 600 When the OD value reaches 30-40, add 5g / L lactose at one time and measure the OD value every 2 hours. 600, analyze the contents of carbon source, lactose and products by liquid phase. When the lactose concentration is less than 0.5g / L, start the lactose addition (i.e. feed 2). The initial flow rate is consistent with the lactose consumption rate. When the lactose accumulation is greater than 1g / L, stop adding lactose immediately, and wait until the next point measurement of lactose is less than 0.5g / L before starting again. Stop fermentation when the strain yield no longer increases.

[0120] Detection of the content of each substance in the fermentation broth: take 1 mL of fermentation broth, centrifuge at 10000 rpm for 10 min, take the supernatant, filter the supernatant with a 0.22 μm water filter, and determine the extracellular concentrations of LNnT, LNT II, ​​neohexaose, lactose and glycerol by HPLC.

[0121] The production of LNnT, LNT II and new hexasaccharide of KB-027 strain was detected by sampling results during 5L batch fed fermentation. 600 represents bacterial concentration) and the yields of LNnT, LNT II, ​​and new hexasaccharides. Figure 1 shown.

[0122] Depend on Figure 1 It can be seen that the yield of LNnT in 5L batch fed fermentation culture is 12.18g / L, of which the byproduct LNT II is 0.34g / L and the new hexasaccharide is 8.03g / L. The results show that both the product and the byproduct increase with the extension of fermentation time, and the new hexasaccharide accounts for a high proportion at the end of fermentation.

[0123] Example 3 Comparison of fermentation of LNnT genetically engineered bacteria with different transporter proteins

[0124] In order to further optimize the fermentation of LNnT genetic engineering bacteria, this embodiment screened 9 transport proteins from the library (as shown in Table 2), and the KB-027 engineering bacteria constructed in Example 2 was used as the starting strain. The endogenous mdfA transport protein was integrated into the hlyE site using the above-mentioned CRISPR-Cpf1-assisted homologous recombination method to construct strain KB-029. The same method was used to integrate different exogenous transport proteins SfSET, EMFST, CaMFST, uSSET, GqSET, KpSET, uSMFST, and SoMFST genes at the hlyE site of the KB-027 strain to obtain eight engineering strains. The constructed strains were named KB-030, KB-031, KB-032, KB-033, KB-034, KB-035, KB-036, and KB-037. The primers involved are shown in Table 2, and the specific construction method is the same as in Example 1.

[0125] Table 2 Construction primers used in the transport gene integration example

[0126]

[0127]

[0128] The strains KB-029-KB-037 were subjected to 5 L fed-batch fermentation. The specific culture method was the same as in Example 2. The extracellular concentrations of LNnT, LNT II, ​​and neohexasaccharide were determined by HPLC. The OD values ​​of the strains were compared. 600 The results are shown in Table 3. Figure 2 .

[0129] Table 3 KB-029-KB-037 engineering bacteria 5L batch fed fermentation results data

[0130]

[0131] It is known that the 5L fed-batch fermentation yield of KB-027 is 12.18 g / L. Figure 2 The data showed that the yields of the KB-030-KB-037 strains constructed after the selected 8 exogenous transporters were integrated into the KB-027 bacterial genome were all improved, and the content of new hexasaccharides was significantly reduced. Among them, KB-037 (integrated the SoMFST transporter protein on the basis of KB-027) performed best, with an LNnT yield of 31.96 g / L, LNT II of 1.31 g / L, and new hexasaccharides of 0.02 g / L.

[0132] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. The use of a transporter in increasing the yield and / or purity of lactose-N-neotetraose, characterized in that: The transporter protein includes at least any one or more of the following A1)-A8): A1) Transporter from Serratia fonticola; A2) Transporter from Escherichia fergusonii; A3) Transporter from Chimaeribacter californicus; A4) Transporter derived from unclassified Serratia (MFS transporter); A5) Transporter from Gibbsiella quercinecans; A6) Transporter from Kosakonia pseudosacchari; A7) unclassified Serratia (sugar efflux transporter) derived transporter; A8) Transporter from Serratia oryzae.

2. Biomaterial, characterized in that The biological material comprises at least one of the following B1)-B3): B1) a nucleic acid molecule encoding the transporter protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) and / or a recombinant vector containing the expression cassette described in B2).

3. The biomaterial according to claim 2, characterized in that: The nucleic acid molecule described in B1) includes any one or more of C1) to C9): C1) the nucleotide sequence comprises the nucleic acid molecule shown in SEQ ID NO.9; C2) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO.11; C3) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO.13; C4) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO.15; C5) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO.17; C6) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO.19; C7) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO.21; C8) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO.23; C9) A nucleic acid molecule having 80% or more identity with the nucleic acid molecule of any one of C1) to C8).

4. A recombinant microorganism capable of secreting and expressing lactose-N-neotetraose, characterized in that: The recombinant microorganism includes any one or more of the following D1)-D4): D1) A recombinant microorganism containing the transporter protein according to claim 1; D2) a recombinant microorganism containing the nucleic acid molecule described in B1) of claim 2; D3) A recombinant microorganism containing the expression cassette described in B2) of claim 2; D4) A recombinant microorganism containing the recombinant vector described in B3) of claim 2.

5. The recombinant microorganism according to claim 4, characterized in that The recombinant microorganism comprises any one of the following E1)-E2): E1) Escherichia bacteria; E2) Escherichia coli.

6. A whole-cell catalyst for preparing lactose-N-neotetraose, characterized in that: The whole-cell catalyst comprises the recombinant microorganism according to claim 4 or 5.

7. A method for producing lactose-N-neotetraose, characterized in that: The method comprises fermenting and producing lactose-N-neotetraose using the recombinant microorganism according to claim 4 or 5 or the whole cell catalyst according to claim 6.

8. Use of the biomaterial according to claim 2 or 3, the recombinant microorganism according to claim 4 or 5, the whole-cell catalyst according to claim 6 or the method according to claim 7 in increasing the yield of lactose-N-neotetraose.

9. Use of the biomaterial according to claim 2 or 3, the recombinant microorganism according to claim 4 or 5, the whole cell catalyst according to claim 6 or the method according to claim 7 in reducing by-products in lactose-N-neotetraose products.

10. Use of the biomaterial according to claim 2 or 3, the recombinant microorganism according to claim 4 or 5, the whole cell catalyst according to claim 6 or the method according to claim 7 in reducing lactose-N-triose and / or lactose-N-neohexose in a lactose-N-neotetraose product.

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