Method for fermentation production of human milk oligosaccharide by cell synthesis method

Abstract

The invention relates to the technical field of bioengineering, in particular to a method for producing human milk oligosaccharide by fermentation through a cell synthesis method. According to the method for producing the human milk oligosaccharide through fermentation with the cell synthesis method, in the fermentation production process of the human milk oligosaccharide, lactose hydrolysate is used for replacing a traditional chemical inducer IPTG to induce expression of synthetic genes of the human milk oligosaccharide, and then synthesis of the human milk oligosaccharide is achieved; the method can avoid the use of a chemical inducer in the production process of the human milk oligosaccharide, effectively solves the food safety problem and the production cost problem caused by the use of the chemical inducer, promotes the realization of green and efficient production of the human milk oligosaccharide, reduces the fermentation production cost, and has a better application prospect.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a method for producing human milk oligosaccharides by fermentation using a cell synthesis method. Background Art

[0002] Human milk oligosaccharides (HMOs) are an important class of bioactive components in breast milk. They are complex oligosaccharides based on lactose and modified with monosaccharides such as fucose, N-acetylglucosamine, and / or sialic acid. Their content in breast milk is second only to lactose and fat. They regulate intestinal health, enhance immunity, and promote brain and nerve development. They have broad application prospects in infant formula, nutritional supplements, maternal and child health products, and food additives. HMOs are synthesized primarily through chemical synthesis, enzymatic synthesis, and cell-based synthesis. Currently, cell-based synthesis is the mainstream method for the synthesis of HMOs, and several HMO products produced through cell-based synthesis are approved for inclusion in infant formula.

[0003] The cellular synthesis of HMOs typically requires inducing the expression of exogenous genes required for HMO synthesis. Currently, the commonly used inducer is isopropyl-β-D-thiogalactopyranoside (IPTG). However, IPTG is a chemical inducer with certain biotoxicity, which hinders its application in the food industry. Furthermore, the high price of IPTG increases the production cost of HMOs. Therefore, the development of green and efficient induction methods that can replace IPTG in the cellular synthesis of HMOs is of great significance for the biosynthesis and application of HMOs. Summary of the Invention

[0004] The present invention provides a method for producing human milk oligosaccharides by fermentation using a cell synthesis method.

[0005] Specifically, the present invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides a method for producing human milk oligosaccharides by fermentation using a cell synthesis method, the method comprising the step of using a lactose hydrolyzate as an inducer to induce the expression of a synthetic gene for human milk oligosaccharides during the fermentation process; wherein the synthetic gene for human milk oligosaccharides initiates transcription with an inducible promoter regulated by LacI protein repression; and the lactose hydrolyzate contains D-galactose.

[0007] Optionally, the lactose hydrolyzate is obtained by hydrolyzing lactose with lactase.

[0008] In the present invention, the inducible promoter regulated by LacI protein repression includes at least one selected from T7-lac promoter or variants thereof, lac promoter or variants thereof, tac promoter or variants thereof.

[0009] In the above induction, the amount of lactose hydrolyzate added is calculated based on the D-galactose contained therein, and the final concentration is 0.01-10 g / L; preferably 0.01-6 g / L.

[0010] In the above-mentioned induction, the volume in the final concentration in the amount added refers to the volume at the time of induction.

[0011] The above-mentioned induction temperature is 25-35°C.

[0012] In the above induction, the lactose hydrolyzate is added to the fermentation system when the growth of the fermentation bacteria is in the late logarithmic phase to the first 1 / 3 stage of the stable phase. Preferably, it is added to the fermentation system in the first 1 / 6 stage of the stable phase. More preferably, it is added to the fermentation system when entering the stable phase. Those skilled in the art can adjust the fermentation temperature by fermentation time, OD 600 The time of stable period can be determined by the consumption of dissolved oxygen and carbon source. For example, the OD value of the fermentation tank can be determined after inoculation and fermentation for 18 hours. 600 When the concentration reaches 140, add it to the fermentation system; you can also observe the dissolved oxygen curve and add it when the dissolved oxygen decreases rapidly and stabilizes after supplementing with carbon source.

[0013] In the present invention, the human milk oligosaccharides include those modified with fucose at the lactose terminus, preferably including one or more of 2'-fucosyllactose, 3'-fucosyllactose, and difucosyllactose; those modified with sialic acid at the lactose terminus, preferably including one or more of 3'-sialyllactose and 6'-sialyllactose; and those modified with N-acetylglucosamine and galactose at the lactose terminus, preferably including one or more of lacto-N-triose II, lacto-N-tetraose, lacto-N-neotetraose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose IV, lacto-N-fucopentaose V, and p-lactose-N-neohexaose.

[0014] In the present invention, the synthesis gene of human milk oligosaccharides includes at least one selected from glycosyltransferase encoding gene, sialic acid synthetase encoding gene, CMP-N-acetylneuraminic acid synthase encoding gene, UDP-GlcNAc2-exoamidase encoding gene, lacY gene, prs gene, glmU gene, glmM gene, glmS gene, pgm gene, galU gene, galE gene, and gtB gene.

[0015] Optionally, the glycosyltransferase comprises at least one selected from the group consisting of sialyltransferase, fucosyltransferase, galactosyltransferase, glucosyltransferase, N-acetylglucosyltransferase and N-acetylgalactosyltransferase.

[0016] In the present invention, the fermentation strain includes one or more genes selected from the group consisting of wcaJ gene, lacZ gene, fucIK gene, nanAKET gene, nudD gene, lacA gene, nagB gene, gcd gene, ugd gene, wecB gene and setA gene, which are partially or completely inactivated.

[0017] Optionally, the bacterial species is bacteria or fungi.

[0018] Optionally, the fermentation uses lactose as a substrate for the synthesis of human milk oligosaccharides.

[0019] In a second aspect, the present invention provides a method for producing a dairy product, comprising the following steps: Prepare human milk oligosaccharides using the method described in the first aspect above; and The human milk oligosaccharide is used to prepare a dairy product.

[0020] In a third aspect, the present invention provides the use of lactose hydrolyzate as an inducer to induce the expression of a target gene; wherein the target gene is transcribed by an inducible promoter regulated by LacI protein repression.

[0021] Preferably, the target gene is a human milk oligosaccharide synthesis gene. The application includes: inducing the expression of the human milk oligosaccharide synthesis gene to produce human milk oligosaccharides.

[0022] The beneficial effects of the present invention include at least: the present invention provides a method for fermenting and producing human milk oligosaccharides using lactose hydrolyzate as an inducer. This method utilizes lactose hydrolyzate to replace the traditional chemical inducer IPTG to induce the expression of human milk oligosaccharide synthesis genes during the fermentation production of human milk oligosaccharides, thereby achieving the synthesis of human milk oligosaccharides. This can avoid the use of chemical inducers in the production of human milk oligosaccharides, effectively solve the food safety and production cost problems caused by the use of chemical inducers, thereby promoting the green and efficient production of human milk oligosaccharides while reducing fermentation production costs. DETAILED DESCRIPTION

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in the technical field of this application. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or can be prepared by existing methods.

[0024] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0025] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0026] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0027] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0028] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional values ​​within the numerical interval is considered continuous and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two numerical endpoints of the numerical range, as well as every integer between the two numerical endpoints. In this document, this is equivalent to directly listing each integer. For example, if n is an integer selected from 1-10, it means that n is any integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges may be combined. In other words, unless otherwise specified, ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0029] In this application, the terms "first," "second," and "third," etc., in "the first aspect," "the second aspect," "the third aspect," etc., are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first," "second," and "third," etc., are only used for non-exhaustive enumeration and description purposes and should be understood not to constitute closed-ended limitations on quantity.

[0030] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0031] All documents mentioned in this application are cited as references in this application, just as each document is cited as a reference individually. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0032] In this application, "homology" is used interchangeably with "identity" and "similarity" and "homology," "identity," and "similarity" refer to the fact that two or more sequences have the same base or amino acid residue at a specific percentage of corresponding positions when the pairwise relationship is maximized in comparison and alignment. The identity of nucleotide sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. Percent homology or identity can be determined using any suitable software program known in the art, for example, as described in Current Protocols in Molecular Biology (FM Ausubel, et al, (eds) 1987, Supplement 30, section 7.7.18). Optional programs include the GCGPileup program, example FASTA (Pearson, et al, 1988, Proc. Natl. Acad. Sci. USA 85: 2444-2448) and BLAST (BLASTanual, Altschul et al. Natl. Cent Biotechnol. linf. NatLibMed. (NCIB NLM NIH), Bethesda, Md.. and Altschul et al, 1997, NAR 25: 3389-3402). Another optional alignment program is ALIGN Plus (Scientific and Educational Software, PA), which preferably uses basic parameters. Another optional sequence software program that can be used here is the TFASTA data search program used in the sequence software package Sequence Software Package version 6.0 (Genetics Computer Group, University of Wisconsin, Madison W). Examples of commonly used sequence alignment algorithms include but are not limited to CLUSTAL Omega, EMBOSS Needle, MAFFT or MUSCLE.

[0033] For the synthesis of HMOs by cell fermentation, current laboratory studies have all chosen IPTG inducers that cannot be degraded by strains. However, in the face of industrial production, the traditional chemical inducer IPTG has certain biological toxicity and is expensive to use, making it unsuitable for the fermentation production of HMOs. There are schemes in the prior art that use lactose to directly replace IPTG, for example, using lactose as an inducer for the expression of recombinant proteins, but these schemes are usually limited to situations where lactose does not participate in the synthesis and metabolism of the target product. However, in contrast, during the synthesis of HMOs, lactose itself participates in the synthesis of HMOs as a precursor substance. Using lactose as an inducer is likely to have an adverse effect on the metabolism and induction effect of the strain, which is not conducive to increasing the yield of HMOs.

[0034] To address the above issues, the applicants of this application have discovered through research that adding lactose hydrolyzate as an inducer for the expression of HMO synthesis genes during the culture process can effectively promote the green and efficient synthesis of HMOs. Lactose hydrolyzate uses lactase (systematic name: β-galactosidase) to hydrolyze lactose into glucose and D-galactose. D-galactose can bind to the intracellular repressor protein encoded by lacI, causing a conformational change in the repressor protein, preventing it from binding to the lacO region upstream of the target gene. This releases its repressive effect on the target gene (and, when using the T7-lac promoter, also releases the repression of the T7 RNA polymerase gene), allowing normal transcription of the target gene and achieving normal expression of the target protein. In fact, D-galactose is also a minor component in the synthesis of HMOs, potentially adversely affecting strain metabolism and HMO production. The applicant unexpectedly discovered that some HMOs exhibited more surprising results when induced with lactose hydrolyzate than with other inducer alternatives; for example, HMO yields with lactose hydrolyzate were significantly superior to those with lactose and D-galactose. Furthermore, the applicant explored the characteristics of different HMOs under non-IPTG induction conditions and, by optimizing the addition dosage, overcame the effects of the inducer on yield, effectively replacing IPTG induction.

[0035] In a first aspect of the present application, a method for producing human milk oligosaccharides by fermentation using a cell synthesis method is provided, the method comprising the step of using a lactose hydrolyzate as an inducer to induce the expression of a synthetic gene for human milk oligosaccharides during the fermentation process; wherein the synthetic gene for human milk oligosaccharides is transcribed using an inducible promoter regulated by LacI protein repression; and the lactose hydrolyzate contains D-galactose.

[0036] In this application, the cell synthesis method refers to the synthesis of HMOs using enzymes produced within biological cells. The biological cells are preferably microbial cells. During the fermentation production of HMOs using the cell synthesis method, cells capable of synthesizing HMOs are fermented and cultured to allow the cells to synthesize HMOs.

[0037] During the fermentation and culturing process, lactose hydrolyzate is used as an inducer to induce the expression of human milk oligosaccharide synthesis genes.

[0038] Lactose is a disaccharide composed of one glucose molecule and one galactose molecule linked by a β-1,4-glycosidic bond. Lactose hydrolysate is obtained by hydrolyzing lactose into glucose and D-galactose using lactase (β-galactosidase) or other enzymes capable of hydrolyzing β-1,4-glycosidic bonds.

[0039] In some embodiments, the lactose hydrolyzate is obtained by hydrolyzing lactose with lactase.

[0040] D-galactose in lactose hydrolyzate is an effector molecule that binds to LacI protein and relieves its repression. Therefore, the amount of lactose hydrolyzate added as an inducer can be calculated according to the content and final concentration of D-galactose therein.

[0041] In some embodiments, during the induction, the amount of lactose hydrolyzate added is calculated based on the D-galactose contained therein, and the final concentration is 0.01-10 g / L; preferably 0.01-6 g / L.

[0042] In some embodiments, the human milk oligosaccharide is LNT, and during the induction, the amount of lactose hydrolyzate added is calculated based on the D-galactose contained therein, and the final concentration is 0.01-2 g / L; preferably 0.01-1.5 g / L; more preferably 0.05-1.5 g / L; more preferably 0.05-0.29 g / L.

[0043] In some embodiments, the human milk oligosaccharide is LNnT, and during the induction, the amount of lactose hydrolyzate added is calculated based on the D-galactose contained therein, and the final concentration is 0.01-3 g / L; preferably 0.01-1.5 g / L; more preferably 0.014-0.3 g / L; more preferably 0.05-0.15 g / L.

[0044] In some embodiments, the human milk oligosaccharide is 3'-SL, and during the induction, the amount of lactose hydrolyzate added is calculated based on the D-galactose contained therein, and the final concentration is 0.1-10 g / L; preferably 0.1-6 g / L; more preferably 1-6 g / L; more preferably 1-3 g / L.

[0045] In some embodiments, the human milk oligosaccharide is 2'-FL, and during the induction, the amount of lactose hydrolyzate added is calculated based on the D-galactose contained therein, and the final concentration is 0.1-10 g / L; preferably 0.1-6 g / L; more preferably 0.14-3 g / L; more preferably 0.14-1.5 g / L.

[0046] The above-mentioned final concentration refers to the final concentration of D-galactose in the fermentation system during induction, ie, the working concentration.

[0047] In some embodiments, the lactose hydrolyzate comprises D-galactose and glucose.

[0048] In some embodiments, the method for preparing the lactose hydrolyzate comprises the following steps: adjusting the pH of a lactose aqueous solution (preferably at a concentration of 10%-30%) to 4.0-5.0, adding lactase at a mass ratio of 1%-2%, performing enzymatic hydrolysis at 50-58°C for 10-20 hours, and then sterilizing and inactivating the enzyme (e.g., at 110-120°C for 15-25 minutes). Those skilled in the art will appreciate that the reaction conditions for lactase can be adjusted according to the manufacturer's guidelines.

[0049] In some embodiments, the inducible promoter regulated by LacI protein repression comprises a lac operator element. The lac operator element includes the lacI gene (encoding the lactose operon repressor protein LacI) and the lacO sequence (the operator element of the lactose operon). The LacI protein can bind to the lacO sequence, thereby inhibiting the activity of the inducible promoter and preventing transcription of downstream genes.

[0050] In this application, the mechanism by which lactose hydrolyzate achieves induction is that the D-galactose contained therein can bind to the LacI protein and change its conformation, causing it to dissociate from the lacO sequence, thereby relieving the inhibition of the promoter and initiating gene expression. Therefore, in principle, the inducible promoter described in this application only needs to contain the lac operator element and be regulated by the LacI protein repression. There are no restrictions on the remaining elements or regions of the promoter.

[0051] It is understandable that in order to achieve a higher expression level of the gene for synthesizing human milk oligosaccharides, the promoter should have stronger transcription initiation activity.

[0052] In some embodiments, the inducible promoter regulated by LacI protein repression includes at least one selected from T7-lac promoter or variants thereof, lac promoter or variants thereof, tac promoter or variants thereof.

[0053] Among them, the T7-lac promoter includes the T7 promoter and the lac operator element. The T7 promoter depends on T7 RNA polymerase for transcription. The lac operator element includes the lacI gene and the lacO sequence.

[0054] The variant of the above promoter refers to a mutant promoter obtained by deletion, insertion or substitution of one or more nucleotides in the above promoter. These mutant promoters can increase or decrease the transcriptional activity, but are still inducible promoters regulated by LacI protein repression.

[0055] Optionally, the variant has at least 80% homology to the promoter, wherein the homology is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%.

[0056] In one embodiment, the T7-lac promoter is connected to the 5' end of the exogenous gene required for HMOs synthesis to initiate transcription of the exogenous gene.

[0057] In one embodiment, the induction temperature is 25-35° C. The induction temperature may vary depending on the suitable temperature of the bacteria used for fermentation. For Escherichia coli, the more suitable induction temperature is 28-32° C.

[0058] In one embodiment, the lactose hydrolyzate is added at a time when the OD of the fermentation strain is 600 When the OD of the fermentation bacteria reaches 0.3 or above; 600 When the OD of the fermentation bacteria reaches 0.5 or above; more preferably 600 When it reaches 0.5-1.0; the most preferred time to add lactose hydrolysis is when the fermentation bacteria are inoculated into the fermentation medium and grow to OD 600 =0.6-0.8.

[0059] In some embodiments, the human milk oligosaccharides include those selected from lactose end modified with fucose, including one or more of 2'-fucosyllactose, 3'-fucosyllactose, and difucosyllactose; lactose end modified with sialic acid, including one or more of 3'-sialyllactose and 6'-sialyllactose; lactose end modified with N-acetylglucosamine and galactose, including one or more of lactose-N-triose II, lactose-N-tetraose, lactose-N-neotetraose, lactose-N-fucopentaose I, lactose-N-fucopentaose II, lactose-N-fucopentaose III, lactose-N-fucopentaose IV, lactose-N-fucopentaose V, and p-lactose-N-neohexaose.

[0060] In this application, human milk oligosaccharide synthesis genes refer to genes required for human milk oligosaccharide synthesis, including genes encoding enzymes that directly catalyze human milk oligosaccharide synthesis and genes encoding synthases of human milk oligosaccharide synthesis precursors. Human milk oligosaccharide synthesis genes can be endogenous genes of the host cell or exogenous genes.

[0061] In some embodiments, the synthesis genes of human milk oligosaccharides include at least one selected from glycosyltransferase encoding genes, sialic acid synthetase encoding genes, CMP-N-acetylneuraminic acid synthase encoding genes, UDP-GlcNAc2-exoamidase encoding genes, lacY genes, prs genes, glmU genes, glmM genes, glmS genes, pgm genes, galU genes, galE genes, and gtB genes.

[0062] In some specific embodiments, the glycosyltransferase comprises at least one selected from sialyltransferase, fucosyltransferase, galactosyltransferase, glucosyltransferase, N-acetylglucosyltransferase, and N-acetylgalactosyltransferase. As an example, the glycosyltransferase is an exogenous gene.

[0063] In some embodiments, the sialyltransferase comprises one or more of α-2,3 sialyltransferase and α-2,6 sialyltransferase.

[0064] The present application has no particular limitation on the source of α-2,3 sialyltransferase, and a suitable α-2,3 sialyltransferase can be selected as needed. As an example, the α-2,3 sialyltransferase is encoded by the ST gene.

[0065] In some embodiments, the amino acid sequence of α-2,3 sialyltransferase is as shown in SEQ ID NO. 1, or has at least 80% homology to the sequence shown in SEQ ID NO. 1, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9% homology.

[0066] It is understood that when human milk oligosaccharides include 3'-sialyllactose (3'-SL) or 6'-sialyllactose (6'-SL), the synthesis genes of human milk oligosaccharides include sialic acid synthase, CMP-N-acetylneuraminic acid synthase and UDP-GlcNAc2-exoamidase in addition to sialyltransferase.

[0067] The present application does not particularly limit the sources of sialic acid synthase, CMP-N-acetylneuraminic acid synthase, and UDP-GlcNAc2-exoamidase. Suitable sialic acid synthase, CMP-N-acetylneuraminic acid synthase, and UDP-GlcNAc2-exoamidase can be selected as needed. As an example, sialic acid synthase, CMP-N-acetylneuraminic acid synthase, and UDP-GlcNAc2-exoamidase are encoded by the neuBCA gene.

[0068] In some specific embodiments, the amino acid sequences of sialic acid synthase, UDP-GlcNAc2-exoamidase, and CMP-N-acetylneuraminic acid synthase are shown in SEQ ID NOs. 2, 3, and 4, respectively, or have at least 80% homology to the sequences shown in SEQ ID NOs. 2, 3, and 4. For example, they have 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9% homology.

[0069] In some specific embodiments, the fucosyltransferase comprises one or more of α-1,2-fucosyltransferase, α-1,3-fucosyltransferase, α-1,3(4)-fucosyltransferase, and α-1,4-fucosyltransferase.

[0070] The present application has no particular limitation on the source of the α-1,2-fucosyltransferase, and a suitable α-1,2-fucosyltransferase can be selected as needed. As an example, the α-1,2-fucosyltransferase is encoded by the Wbgl gene of Escherichia coli O126.

[0071] In some specific embodiments, the amino acid sequence of the α-1,2-fucosyltransferase is as shown in SEQ ID NO. 5, or has at least 80% homology to the sequence shown in SEQ ID NO. 5, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 99.9% homology.

[0072] In some specific embodiments, the galactosyltransferase comprises one or more of β-1,3-galactosyltransferase and 1,4-galactosyltransferase.

[0073] The present application has no particular limitation on the source of β-1,3-galactosyltransferase, and a suitable β-1,3-galactosyltransferase can be selected as needed. As an example, β-1,3-galactosyltransferase is encoded by the SewbdO gene.

[0074] In some specific embodiments, the amino acid sequence of β-1,3-galactosyltransferase is as shown in SEQ ID NO. 6, or has at least 80% homology to the sequence shown in SEQ ID NO. 6, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 99.9% homology.

[0075] The present application has no particular limitation on the source of β-1,4-galactosyltransferase, and a suitable β-1,4-galactosyltransferase can be selected as needed. As an example, β-1,4-galactosyltransferase is encoded by the LgtB gene from Helicobacter pylori.

[0076] In some specific embodiments, the amino acid sequence of β-1,4-galactosyltransferase is as shown in SEQ ID NO. 7, or has at least 80% homology to the sequence shown in SEQ ID NO. 7, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 99.9% homology.

[0077] In some specific embodiments, the glucosyltransferase comprises one or more of α-glucosyltransferase, β-1,2-glucosyltransferase, β-1,3-glucosyltransferase, and β-1,4-glucosyltransferase.

[0078] In some specific embodiments, the N-acetylglucosyltransferase comprises one or more of β-1,3-N-acetylglucosaminyltransferase and β-1,6-N-acetylglucosaminyltransferase.

[0079] The present application does not particularly limit the source of β-1,3-N-acetylglucosamine transferase, and a suitable β-1,3-N-acetylglucosamine transferase can be selected as needed. As an example, β-1,3-N-acetylglucosamine transferase is encoded by the LgtA gene from Neisseria meningitidis MC58.

[0080] In some specific embodiments, the amino acid sequence of β-1,3-N-acetylglucosamine transferase is as shown in SEQ ID NO. 8, or has at least 80% homology to the sequence shown in SEQ ID NO. 8, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9% homology.

[0081] In some embodiments, the N-acetylgalactosyltransferase comprises α-1,3-N-acetylgalactosaminyltransferase.

[0082] In some specific embodiments, the human milk oligosaccharide includes 3'-SL, and the synthesis genes of the human milk oligosaccharide include genes encoding sialic acid synthase, CMP-N-acetylneuraminic acid synthase, UDP-GlcNAc2-exoamidase, and α-2,3 sialyltransferase.

[0083] In some specific embodiments, the human milk oligosaccharide includes 3'-SL, and the synthesis genes of the human milk oligosaccharide include neuBCA and ST genes.

[0084] In some specific embodiments, the human milk oligosaccharide comprises LNT, and the synthesis genes of the human milk oligosaccharide comprise genes encoding β-1,3-N-acetylglucosaminyltransferase and β-1,3-galactosyltransferase.

[0085] In some specific embodiments, the human milk oligosaccharide includes LNT, and the synthesis genes of the human milk oligosaccharide include LgtA and SewbdO.

[0086] In some specific embodiments, the human milk oligosaccharide comprises LNnT, and the synthesis genes of the human milk oligosaccharide comprise genes encoding β-1,3-N-acetylglucosaminyltransferase and β-1,4-galactosyltransferase.

[0087] In some specific embodiments, the human milk oligosaccharide includes LNnT, and the synthesis genes of the human milk oligosaccharide include LgtA and LgtB.

[0088] In some specific embodiments, the human milk oligosaccharide includes 2'-FL, and the genes for synthesizing human milk oligosaccharides include α-1,2-fucosyltransferase, mannose-1-phosphate guanosyltransferase, phosphomannose mutase, GDP-mannose-4,6-dehydratase and GDP-L-fucose synthase.

[0089] In some specific embodiments, the human milk oligosaccharide includes 2'-FL, and the genes for synthesizing human milk oligosaccharides include CBGW derived from Escherichia coli K12 and Wbgl derived from Escherichia coli O126; wherein, CBGW includes manB (encoding protein as shown in SEQ ID NO.9), manC (encoding protein as shown in SEQ ID NO.10), gmd (encoding protein as shown in SEQ ID NO.11) and wcaG (encoding protein as shown in SEQ ID NO.12).

[0090] In addition, when the human milk oligosaccharides include 6'-SL, the genes for synthesizing human milk oligosaccharides include an α-2,6 sialyltransferase encoding gene; when the human milk oligosaccharides include 3'-FL, the genes for synthesizing human milk oligosaccharides include an α-2,6 sialyltransferase encoding gene and mannose-1-phosphate guanosine transferase, phosphomannose mutase, GDP-mannose-4,6-dehydratase and GDP-L-fucose synthase.

[0091] In some embodiments, the gene for synthesizing human milk oligosaccharides is inserted into an expression plasmid, for example, pETDuet-1, pCDFDuet-1, pRSFDuet-1, etc.

[0092] In some embodiments, the gene for synthesizing human milk oligosaccharides is inserted downstream of the T7-lac promoter system of the pRSFDuet-1 plasmid.

[0093] In some embodiments, the fermentation strain includes one or more genes selected from the group consisting of wcaJ gene, lacZ gene, fucIK gene, nanAKET gene, nudD gene, lacA gene, nagB gene, gcd gene, ugd gene, wecB gene and setA gene, which are partially or completely inactivated.

[0094] In some embodiments, the fermentation strain includes one or more genes selected from the group consisting of wcaJ gene, lacZ gene, fucIK gene, nanAKET gene, nudD gene, lacA gene, nagB gene, gcd gene, ugd gene, wecB gene and setA gene, which are partially or completely inactivated; and at the same time, the genes for synthesizing human milk oligosaccharides are heterologously expressed; wherein, the genes for synthesizing human milk oligosaccharides are transcribed using an inducible promoter regulated by LacI protein repression.

[0095] In some embodiments, the wcaJ, lacZ and fucIK genes are knocked out in the fermentation strain.

[0096] The bacterial species used for fermentation in this application can be any host cell commonly used in the art, and there are no particular limitations. Host cells include prokaryotic and / or eukaryotic cells. In some optional examples, they can be bacteria, such as Escherichia coli and Bacillus subtilis; in some optional examples, they can be eukaryotic cells, such as yeast cells, plant cells, insect cells, and animal cells, such as mammalian cells (e.g., mouse cells, human cells, etc.). In principle, there are no limitations on bacterial host cells; they can be eubacteria (Gram-positive or Gram-negative) or archaea, as long as they allow for the insertion of the gene of interest and can be cultured on a production scale.

[0097] As an example, the bacteria may be Escherichia coli ( Escherichia coli ), Klebsiella oxytoca ( Klebsiella oxytoca ), Anaerobic spirillum succinate-producing bacteria ( Anaerobiospirillum succiniciproducens ), Actinobacillus succinogenes ( Actinobacillus succinogenes ), Mannheimia succiniciproducens ( Mannheim succinic producing ), Rhizobium vulgaris ( Rhizobium etli ), Bacillus subtilis ( Bacillus subtle ), Bacillus methanolicus ( Methanolic Bacillus ), Corynebacterium glutamicum ( Corynebacterium glutamic ), Gluconobacter oxydans ( Gluconobacter oxidans ), Zymomonas mobilis ( Zymomonas mobile ), Lactococcus lactis ( Lactococcus lactis ), Lactobacillus plantarum ( Lactobacillus plantarum ), Streptomyces coelicolor ( Streptomyces coelicolor ), Clostridium acetobutylicum ( Clostridium acetobutylic acid ), Pseudomonas fluorescens ( Pseudomonas fluorescens ) or Pseudomonas putida ( Pseudomonas putida The yeast or fungus may be Saccharomyces cerevisiae ( Saccharomyces cerevisiae ), Schizosaccharomyces pombe ( Schizosaccharomyces pombe ), Kluyveromyces lactis ( Kluyveromyces lactis ), Kluyveromyces marxianus ( Kluyveromyces marxianus ), Aspergillus terreus ( Aspergillus terreus ), Aspergillus niger ( Aspergillus niger ), Pichia pastoris ( Shepherd's pie )、Rhizopus arrhizus( Rhizopus arrhizal ) or Yarrowia lipolytica ( Yarrowia lipolytica )wait.

[0098] In some embodiments, the strain used for fermentation is Escherichia coli, which may be Escherichia coli BL21 (DE3), or further optionally, Escherichia coli BL21 star (DE3).

[0099] The method for constructing the fermentation strain described above comprises the following steps: introducing into the base plate bacteria an expression cassette containing an exogenous gene required for HMOs synthesis under the control of an inducible promoter regulated by LacI protein repression.

[0100] In some embodiments, the introduction method includes one or more of heat shock method, electroporation method, calcium ion method and spheroplast method.

[0101] It is understood that the expression of the exogenous gene required for HMO synthesis in the host cell can be achieved through genomic integration or expression vector expression, as commonly used in the art, and is not specifically limited in this application. For example, an expression cassette containing the exogenous gene required for HMO synthesis under the control of an inducible promoter regulated by LacI protein repression can be integrated into the host cell genome or cloned into an expression vector such as pETDuet-1, pCDFDuet-1, or pRSFDuet-1.

[0102] In the fermentation process of the present application, lactose is used as a substrate for the synthesis of human milk oligosaccharides. Therefore, the culture medium used in the fermentation includes lactose.

[0103] It is understandable that the present application does not specifically limit the fermentation culture method and the type of culture medium used for fermentation culture, and it can be any method and any culture medium commonly used in the art. As an example, the fermentation culture medium comprises the following components: lactose, a nitrogen source (such as yeast extract), phosphate, ammonium salt, potassium salt, glycerol and magnesium salt. As an example, the fermentation culture medium comprises the following components: lactose (preferably 5-20 g / L) and diammonium hydrogen phosphate (preferably 2-8 g / L), KH2PO4 (preferably 10-16 g / L), citric acid (preferably 0.5-2.5 g / L), yeast extract (preferably 2-6 g / L), glycerol (preferably 15-25 g / L) and MgSO4 heptahydrate (preferably 0.5-2.5 g / L); or, the fermentation medium comprises the following components: peptone (preferably 130-170 g / 20 L), yeast extract powder (preferably 40-60 g / 20 L), (NH4)2SO4 (preferably 80-120 g / 20 L), K2HPO4 (preferably 210-25 0g / 20L), KH2PO4 (preferably 185-225g / 20L), glycerol (preferably 230-270g / 20L), citric acid (preferably 5-10g / 20L), glucose (preferably 220-280g / 20L), MgSO4·7H2O (preferably 40-60g / 20L), anhydrous CaCl2 (preferably 0.4-0.6g / 20L), 1000× vitamin B1 (preferably 20-30mL / 20L), pH 6.6-7.0.

[0104] Optionally, the fermentation temperature is 35°C to 38°C; Optionally, the fermentation time is 50 h to 100 h.

[0105] According to a second aspect of the present application, a method for producing a dairy product is provided, comprising the following steps: Prepare HMOs using the method of the first aspect above; and Preparation of dairy products using HMOs.

[0106] It is understandable that the method for preparing dairy products using HMOs can be any method commonly used in the art, and this application does not impose any particular limitation.

[0107] The third aspect of the present application provides the use of lactose hydrolyzate as an inducer to induce the expression of a target gene; wherein the target gene is transcribed by an inducible promoter regulated by LacI protein repression.

[0108] In the third aspect, the lactose hydrolyzate and the inducible promoter regulated by LacI protein repression are as described in the first aspect.

[0109] In principle, there are no specific restrictions on the target gene described above, as long as the gene is transcribed by an inducible promoter regulated by the LacI protein. As an example, the target gene can be a gene encoding an enzyme involved in metabolite synthesis, a gene encoding a catalytic enzyme, a gene encoding a recombinant protein, a gene encoding a guide RNA or an interfering RNA, etc.

[0110] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0111] The raw materials involved in the following examples, unless otherwise specified, can all be sourced from commercial sources. The instruments used, unless otherwise specified, can all be sourced from commercial sources. The processes involved, unless otherwise specified, are all routinely selected by those skilled in the art.

[0112] The preparation method of the 20% lactose hydrolyzate used in the following examples is as follows: (1) Prepare 20% lactose aqueous solution: weigh 20 g lactose, dilute to 100 mL with distilled water, and adjust the pH to 4.5 with dilute hydrochloric acid (2 mol / L); (2) Enzymatic hydrolysis: Weigh 0.24 g of lactase and add it to a 20% lactose aqueous solution. Dissolve it and then heat and stir (55°C, 16 h) to completely hydrolyze the lactose into glucose and D-galactose. (3) Sterilization and enzyme inactivation: After the enzymatic hydrolysis in step (2) is completed, the hydrolyzate is subjected to high temperature treatment (115°C, 20 min).

[0113] In the above method, the conditions for completely hydrolyzing lactose are within the common technical knowledge of those skilled in the art and can be adjusted as needed, as long as lactose is completely hydrolyzed into glucose and D-galactose.

[0114] The HMO-producing strains demonstrated in the following examples utilize simplified chassis cell modifications, primarily for the purpose of producing a variety of HMOs. This ensures excellent stability during subsequent passage and application. To further increase HMO production, conventional methods in the art can be employed to optimize metabolic fluxes that favor HMO synthesis, based on the genetically engineered strains used in the following examples. The production strains in the examples are provided for illustrative purposes only and should not be construed as limiting HMO-producing strains.

[0115] In the following examples, Escherichia coli BL21 star (DE3) was used as the recipient strain, and the wcaJ, lacZ, and fucIK genes in the recipient strain genome were knocked out using the λ-Red homologous recombination system to obtain chassis cells. The E. coli chassis cells used in the following examples were all this chassis cells.

[0116] In the following examples, HMOs were detected using the following methods: 2'-FL: the method described in patent application CN119534680A; 3'-SL: the high-performance liquid chromatography column-ultraviolet detector (HPLC-UV) analysis method for 3'-SL described in the "Public Solicitation of Opinions on Six New Food Additives, Including Peroxidase," issued by the National Food Safety Risk Assessment Center on October 24, 2024; LNnT: T / STIC 130021-2024; LNT: the LNT detection method described in the "Public Solicitation of Opinions on Eight New Food Additives, Including Aminopeptidase," issued by the National Food Safety Assessment Center on March 21, 2025.

[0117] Example 1 Fermentation production of lactose-N-tetraose (LNT) 1. Construction of lactose-N-tetraose production strain (1) The artificially synthesized lgtA-SewbdO fragment (wherein the amino acid sequence of the lgtA protein is shown in SEQ ID NO. 8, and the amino acid sequence of the SewbdO protein is shown in SEQ ID NO. 6) was cloned into the BamHI / PstI site of pRSFDuet-1 by enzyme digestion and ligation, so that the T7-lac promoter system in the plasmid pRSFDuet-1 was located upstream of the lgtA-SewbdO fragment, thereby obtaining the expression plasmid pRSFDuet-lgtA-SewbdO.

[0118] (2) The plasmid pRSFDuet-lgtA-SewbdO was transformed into Escherichia coli chassis cells by heat shock method, spread on LB plates containing kanamycin, and cultured at 37°C overnight; transformants were randomly picked for colony PCR verification, and positive transformants containing lgtA-SewbdO fragments were screened and sequenced to obtain a lactose-N-tetraose production strain with correct sequencing.

[0119] 2. Fermentation yield verification (1) The lactose-N-tetraose producing strain constructed in 1 above was inoculated into 5 mL of LB medium for activation culture to obtain seed liquid.

[0120] (2) The seed solution (OD 600=1), inoculated into 35 mL of fermentation medium at an inoculum size of 2%, with an initial culture temperature of 37°C and a rotation speed of 250 rpm.

[0121] The formula of the fermentation medium is shown in Table 1.

[0122] Table 1

[0123] (3) Detect bacterial growth in the culture medium to OD 600 =0.6-0.8, different volumes (5 µL, 20 µL, 50 µL, 500 µL) of 20% lactose hydrolyzate were added, the temperature was set at 30°C, and the culture was carried out for 60 h.

[0124] Example 2 Fermentation Production of Lactose-N-neotetraose (LNnT) 1. Construction of lactose-N-neotetraose production strain (1) The artificially synthesized lgtA-lgtB fragment (wherein the amino acid sequence of the lgtA protein is shown in SEQ ID NO. 8, and the amino acid sequence of the lgtB protein is shown in SEQ ID NO. 7) was cloned into the BamHI / PstI site of pRSFDuet-1 by enzyme digestion and ligation, so that the T7-lac promoter system in the plasmid pRSFDuet-1 was located upstream of the lgtA-lgtB fragment, thereby obtaining the expression plasmid pRSFDuet-lgtA-lgtB.

[0125] (2) The plasmid pRSFDuet-lgtA-lgtB was transformed into Escherichia coli chassis cells by heat shock method, and spread on LB plates containing kanamycin and cultured at 37°C overnight; transformants were randomly picked for colony PCR verification, and positive transformants containing lgtA-lgtB fragments were screened and sequenced to obtain a lactose-N-neotetraose production strain with correct sequencing, which was named Escherichia coli ( Escherichia coli )LNnTIA.

[0126] Escherichia coli ( Escherichia coli ) LNnTIA was deposited in the China Center for Type Culture Collection (CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code 430072) on April 2, 2025, and was classified as Escherichia coli Escherichia coli , the deposit number is CCTCC NO: M 2025679.

[0127] 2. Fermentation yield verification The lactose-N-neotetraose producing strain constructed in 1 above was used for a fermentation experiment, and the fermentation method was the same as that in Example 1.

[0128] Example 3 Fermentation Production of 3'-sialyllactose (3'-SL) 1. Construction of 3'-sialyllactose production strain (1) The artificially synthesized neuBCA-ST fragment (wherein the amino acid sequences of sialic acid synthase, UDP-GlcNAc2-exoamidase, and CMP-N-acetylneuraminic acid synthase encoded by neuBCA are shown in SEQ ID NO. 2, 3, and 4, respectively, and the amino acid sequence of the ST-encoded protein is shown in SEQ ID NO. 1) was cloned into the BamHI / PstI site of pRSFDuet-1 by enzyme digestion and ligation, so that the T7-lac promoter system in the plasmid pRSFDuet-1 was located upstream of the neuBCA-ST fragment, thereby obtaining the expression plasmid pRSFDuet-neuBCA-ST.

[0129] (2) The plasmid pRSFDuet-neuBCA-ST was transformed into Escherichia coli chassis cells by heat shock method, and spread on LB plates containing kanamycin and cultured at 37°C overnight; transformants were randomly picked for colony PCR verification, and positive transformants containing neuBCA-ST fragments were screened and sequenced to obtain a 3'-sialyllactose production strain with correct sequencing, which was named Escherichia coli ( Escherichia coli )3'-SL-Ec II(A-1).

[0130] Escherichia coli ( Escherichia coli ) 3'-SL-Ec II (A-1) was deposited in the China Center for Type Culture Collection (CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code 430072) on September 4, 2024, and was classified as Escherichia coli Escherichia coli , the deposit number is CCTCC NO: M 20241909.

[0131] 2. Fermentation yield verification Fermentation experiments were carried out using the 3'-sialyllactose producing strain constructed in 1 above. The fermentation method was different from that in Example 1 except that the amount of 20% lactose hydrolyzate added during the fermentation process was 50 μL, 500 μL, 1 mL, and 2 mL, respectively.

[0132] Example 4 Fermentation Production of 2'-Fucosyllactose (2'-FL) 1. Construction of 2'-fucosyllactose production strain (1) The artificially synthesized CBGW-Wbgl fragment (CBGW includes manB (The encoded protein is shown in SEQ ID NO.9) 、manC(The encoded protein is shown in SEQ ID NO.10) 、gmd (encoded protein as shown in SEQ ID NO.11) and wcaG (the encoded protein is shown in SEQ ID NO.12), and the amino acid sequence of the Wbgl encoded protein is shown in SEQ ID NO.5) was cloned into the BamHI / PstI site of pRSFDuet-1 by enzyme digestion and ligation, so that the T7-lac promoter system in the plasmid pRSFDuet-1 was located upstream of the CBGW-Wbgl fragment, and the expression plasmid pRSFDuet-CBGW-Wbgl was obtained.

[0133] (2) The plasmid pRSFDuet-CBGW-Wbgl was transformed into Escherichia coli chassis cells by heat shock method, and spread on LB plates containing kanamycin and cultured at 37°C overnight; transformants were randomly picked for colony PCR verification, and positive transformants containing CBGW-Wbgl fragments were screened and sequenced to obtain the correctly sequenced 2'-fucosyllactose producing strain, which was named Escherichia coli ( Escherichia coli ) FL-Ec IV.

[0134] Escherichia coli ( Escherichia coli ) FL-Ec IV was deposited in the China Center for Type Culture Collection (CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code 430072) on August 4, 2022, and was classified as Escherichia coli Escherichia coli , the deposit number is CCTCC NO: M 20221238.

[0135] 2. Fermentation yield verification Fermentation experiments were carried out using the 2'-fucosyllactose-producing strain constructed in 1 above. The fermentation method was different from that in Example 1 except that the amount of 20% lactose hydrolyzate added during the fermentation process was 50 μL, 500 μL, 1 mL, and 2 mL, respectively.

[0136] Example 5 LNT fermentation tank fermentation verification 1. The seed solution obtained according to the method of step 2 of Example 1 was inoculated into 200 mL of LB medium at a 1% inoculum size and cultured at 37°C, 250 rpm for 5-6 h to obtain a secondary seed solution.

[0137] 2. The secondary seed liquid obtained in step 1 was transferred to a 50L fermenter containing fermentation medium at a 1% inoculum size. The initial culture temperature was 37°C, the rotation speed was 250 rpm, the ventilation volume was 35 NL / min, the tank pressure was 0.06 MPa, and the dissolved oxygen content was controlled to be greater than 2 mg / L.

[0138] The fermentation medium formula includes: 150 g peptone, 50 g yeast extract powder, 100 g (NH4)2SO4, 230 g K2HPO4, 205 g KH2PO4, 250 g glycerol, 7.5 g citric acid, 5 mL defoamer, 250 g glucose, 50 g MgSO4·7H2O, 0.5 g anhydrous CaCl2, 25 mL 1000× vitamin B1, and H2O to 20 L; the pH value is 6.8.

[0139] 3. When the initial glycerol in the fermentation culture is completely consumed, glycerol is added to the fermenter at a rate of 3.4 g / L / h. The culture temperature is 30°C.

[0140] 4. After 4 hours of carbon source supplementation (18 hours after the start of fermentation), 20% lactose hydrolyzate was added to make the final concentration of the corresponding D-galactose in the fermentation system at this time 0.057 g / L, and lactose was supplemented. The lactose content and consumption were monitored. The amount of sugar supplement was to control the lactose content in the fermentation liquid at 10 g / L. A total of 4 sugar supplements were performed, and the fermentation was terminated after 90 hours.

[0141] Example 6 Fermentation Verification of LNnT in a Fermenter The fermentation tank fermentation verification method of LNnT differs from the fermentation tank fermentation verification method of LNT in Example 5 only in that the seed solution used in step 1 is the seed solution obtained according to the method of step 2 of Example 2, and the amount of 20% lactose hydrolyzate added is such that the final concentration of the corresponding D-galactose in the fermentation system at this time is 0.143 g / L.

[0142] Example 7 Fermentation Verification of LNnT in a Fermenter Fermentation verification was performed using the genetically engineered bacteria constructed in Example 1 of patent application CN119736222A (comprising the pSPIN-N32-lgtA2-lgtB2-vag integration vector and the pET-lacY-galE expression vector, and the setA gene, lacZ gene, fucIK gene and wcaJ gene in the chassis strain were knocked out) and the fermentation verification method in Example 1 of the patent application, with the only difference being that 20% lactose hydrolyzate (the same as in Example 6) was added as an inducer, and IPTG was not added.

[0143] Example 8 Fermentation Verification of 2'-FL The fermentation tank fermentation verification method for 2'-FL differs from the fermentation tank fermentation verification method for LNT in Example 5 only in that the seed solution used in step 1 is the seed solution obtained according to the method of step 2 of Example 4; and the amount of 20% lactose hydrolyzate added is such that the final concentration of the corresponding D-galactose in the fermentation system at this time is 1.43 g / L.

[0144] Comparative Example 1 In this comparative example, the lactose-N-tetraose production strain constructed in step 1 of Example 1 was used for fermentation verification; during the fermentation yield verification of the production strain, an IPTG solution with a concentration of 1 M was added to a final concentration of 0.2 mM (optimal induction concentration) or a 20% lactose solution was added as an inducer, and the remaining fermentation conditions except the inducer were the same as in Example 1.

[0145] Comparative Example 2 This comparative example uses the lactose-N-neotetraose production strain constructed in step 1 of Example 2 for fermentation verification; during the fermentation yield verification of the production strain, an IPTG solution with a concentration of 1 M was added to a final concentration of 0.2 mM as an inducer, and the remaining fermentation conditions except the inducer were the same as in Example 1.

[0146] Comparative Example 3 This comparative example uses the 3'-sialyllactose production strain constructed in step 1 of Example 3 for fermentation verification; during the fermentation yield verification of the production strain, a 1 M IPTG solution was added to a final concentration of 0.2 mM or a 20% lactose solution was added as an inducer, and the remaining fermentation conditions except the inducer were the same as those in Example 1.

[0147] Comparative Example 4 This comparative example uses the 2'-fucosyllactose production strain constructed in step 1 of Example 4 for fermentation verification; during the fermentation yield verification of the production strain, a 1 M IPTG solution was added to a final concentration of 0.2 mM or a 20% lactose solution was added as an inducer, and the remaining fermentation conditions except the inducer were the same as in Example 1.

[0148] Comparative Example 5 The only difference between this comparative example and Example 5 is that in step 4, IPTG (final concentration of 0.2 mM) was used as the inducer instead of D-galactose.

[0149] Comparative Example 6 The only difference between this comparative example and Example 6 is that IPTG (final concentration of 0.2 mM) was used as the inducer instead of D-galactose.

[0150] Comparative Example 7 The only difference between this comparative example and Example 7 is that IPTG (final concentration of 0.2 mM) was used as the inducer instead of D-galactose.

[0151] Comparative Example 8 The only difference between this comparative example and Example 8 is that IPTG (final concentration of 0.2 mM) was used as the inducer instead of D-galactose.

[0152] Fermentation yield analysis of HMOs in experimental example The final product yields of the fermentation yield verification in Example 1 and Comparative Example 1 were tested, and the results are shown in Table 2.

[0153] Table 2

[0154] As shown in Table 2, the fermentation method using 20% ​​lactose hydrolyzate as an inducer in Example 1 can effectively replace the chemical inducer IPTG to induce the expression of LNT synthesis genes, thereby achieving efficient synthesis of LNT; within a certain range, the yield of LNT increases with the increase in the amount of lactose hydrolyzate added.

[0155] The final product yields of the fermentation yield verification in Example 2 and Comparative Example 2 were tested, and the results are shown in Table 3.

[0156] Table 3

[0157] As shown in Table 3, the fermentation method using 20% ​​lactose hydrolyzate as an inducer in Example 2 can effectively replace the chemical inducer IPTG to induce the expression of the LNnT synthetic gene; within a certain range, the yield of LNnT increases with the increase in the amount of lactose hydrolyzate added.

[0158] The final product yields of the fermentation yield verification in Example 3 and Comparative Example 3 were tested, and the results are shown in Table 4.

[0159] Table 4

[0160] As shown in Table 4, the fermentation method using 20% ​​lactose hydrolyzate as an inducer in Example 3 can replace the chemical inducer IPTG to induce the expression of the 3'-SL synthesis gene; within a certain range, the yield of 3'-SL increases with the increase in the amount of lactose hydrolyzate added.

[0161] The final product yields of the fermentation yield verification in Example 4 and Comparative Example 4 were tested, and the results are shown in Table 5.

[0162] Table 5

[0163] As shown in Table 5, the fermentation method using 20% ​​lactose hydrolyzate as an inducer in Example 4 can replace the chemical inducer IPTG to induce the expression of the 2'-FL synthetic gene; within a certain range, the yield of 2'-FL increases with the increase in the amount of lactose hydrolyzate added.

[0164] The final product yields of the fermentation verifications of Example 5 and Comparative Example 5 were tested, and the results showed that the fermentation yield of LNT in Example 5 was increased by 24.23% compared with that in Comparative Example 5.

[0165] The final product yields of the fermentation verifications of Example 6 and Comparative Example 6 were tested, and the results showed that the fermentation yield of LNnT in Example 6 was increased by 44.05% compared with that in Comparative Example 6.

[0166] The final product yields of the fermentation verifications of Example 7 and Comparative Example 7 were tested, and the results showed that the fermentation yield of LNnT in Example 7 was 67.39 g / L, which was increased by 46.51% compared with Comparative Example 7.

[0167] The final product yields of the fermentation verifications of Example 8 and Comparative Example 8 were tested. The results showed that the fermentation yield of 2'-FL in Example 8 was 90.26 g / L, and the fermentation yield in Comparative Example 8 was 93.09 g / L.

[0168] In summary, human milk oligosaccharides containing N-acetylglucosamine and galactose as the core structure at the lactose end can overcome the metabolic production interference caused by induction with lactose hydrolysate; human milk oligosaccharides modified with fucose at the lactose end and human milk oligosaccharides modified with sialic acid at the end can also achieve the replacement of IPTG inducer with lactose hydrolysate.

[0169] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for producing human milk oligosaccharides by fermentation using a cell synthesis method, characterized in that: The method comprises the steps of using lactose hydrolyzate as an inducer to induce the expression of human milk oligosaccharide synthesis genes during fermentation; Wherein, the synthetic gene of human milk oligosaccharide initiates transcription with an inducible promoter regulated by LacI protein repression; The lactose hydrolyzate contains D-galactose.

2. The method according to claim 1, characterized in that The lactose hydrolyzate is obtained by hydrolyzing lactose with lactase.

3. The method according to claim 1 or 2, characterized in that The inducible promoter regulated by LacI protein repression includes at least one selected from T7-lac promoter or variants thereof, lac promoter or variants thereof, tac promoter or variants thereof.

4. The method according to any one of claims 1 to 3, characterized in that During the induction, the amount of lactose hydrolyzate added is calculated based on the D-galactose contained therein, and the final concentration is 0.01-10 g / L, preferably 0.01-6 g / L.

5. The method according to any one of claims 1 to 4, characterized in that The induction temperature is 25-35°C; And / or, the lactose hydrolyzate is added to the fermentation system when the growth of the fermentation bacteria is in the late logarithmic phase to the first 1 / 3 of the stationary phase.

6. The method according to any one of claims 1 to 5, characterized in that The human milk oligosaccharides include those selected from the group consisting of lactose modified with fucose at the end, preferably including one or more of 2'-fucosyllactose, 3'-fucosyllactose, and difucosyllactose; those selected from the group consisting of lactose modified with sialic acid at the end, preferably including one or more of 3'-sialyllactose and 6'-sialyllactose; and those selected from the group consisting of lactose modified with N-acetylglucosamine and galactose at the end, preferably including one or more of lactose-N-triose II, lactose-N-tetraose, lactose-N-neotetraose, lactose-N-fucopentaose I, lactose-N-fucopentaose II, lactose-N-fucopentaose III, lactose-N-fucopentaose IV, lactose-N-fucopentaose V, and p-lactose-N-neohexaose.

7. The method according to any one of claims 1 to 6, characterized in that The human milk oligosaccharide synthesis gene includes at least one selected from the group consisting of a glycosyltransferase encoding gene, a sialic acid synthetase encoding gene, a CMP-N-acetylneuraminic acid synthase encoding gene, a UDP-GlcNAc2-exoamidase encoding gene, a lacY gene, a prs gene, a glmU gene, a glmM gene, a glmS gene, a pgm gene, a galU gene, a galE gene, and a gtB gene; Preferably, the glycosyltransferase comprises at least one selected from the group consisting of sialyltransferase, fucosyltransferase, galactosyltransferase, glucosyltransferase, N-acetylglucosyltransferase and N-acetylgalactosyltransferase.

8. The method according to any one of claims 1 to 7, characterized in that The fermentation strain includes one or more genes selected from the group consisting of wcaJ gene, lacZ gene, fucIK gene, nanAKET gene, nudD gene, lacA gene, nagB gene, gcd gene, ugd gene, wecB gene and setA gene, which are partially or completely inactivated; Preferably, the bacterial species is bacteria or fungi; Preferably, the fermentation uses lactose as a substrate for the synthesis of human milk oligosaccharides.

9. A method for producing a dairy product, characterized in that: The steps include: Human milk oligosaccharides are prepared by the method according to any one of claims 1 to 8; and The human milk oligosaccharide is used to prepare a dairy product.

10. Use of lactose hydrolyzate as an inducer to induce target gene expression; wherein, The target gene is transcribed by an inducible promoter regulated by LacI protein repression.

Images