Application of protein in construction of engineering bacteria for producing 6 '-sialyllactose

Abstract

The invention provides application of protein in construction of engineering bacteria for producing 6 '-sialic acid lactose, and relates to the technical field of microbial genetic engineering. According to the invention, new alpha2, 6-sialyltransferase with the potential of producing 6 '-sialyllactose is obtained through screening, and genes for coding precursor CMP-Neu5Ac synthetic pathway enzyme and alpha2, 6-sialyltransferase are introduced into escherichia coli to construct a new biosynthesis pathway of 6'-sialyllactose so as to construct engineering bacteria for efficiently producing 6 '-sialyllactose. Besides, a precursor CMP-Neu5Ac synthetic pathway enzyme and alpha2, 6-sialyltransferase are integrated and expressed in the engineering bacteria, so that the instability problem caused by plasmids is avoided, meanwhile, the dependence on antibiotics in the production process is reduced, and the environmental risk in the production process is reduced.

Description

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on November 18, 2024, with application number "202411646735.1" and invention name "Application of protein in constructing engineered bacteria producing 6'-sialyllactose", the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application relates to the technical field of microbial genetic engineering, and in particular to the application of proteins in constructing engineered bacteria that produce 6'-sialyllactose. Background Art

[0003] Human milk oligosaccharides (HMOs) are the third most abundant solid component in human milk. They are complex and diverse soluble sugars derived from monosaccharides such as glucose (Glc), galactose (Gal), N-acetylglucosamine (GlcNAc), fucose (Fuc), or sialic acid (Neu5Ac), constituting non-fucosylated neutral, fucosylated, and sialylated HMOs. 6′-sialyllactose (6′-SL, Neu5Acα2,6Galβ1,4Glc) is the simplest sialylated HMO containing a terminal sialic acid. It is formed by a Neu5Ac molecule linked to the galactosyl group of lactose via an α2,6-glycosidic bond.

[0004] 6′-sialyllactose has demonstrated its diverse biological activities and potential health benefits in biomedical research. It has been shown to play an important role in regulating the intestinal microbiome, enhancing immune responses, combating viral infections, preventing neonatal necrotizing enterocolitis (NEC), and promoting neurodevelopment and cognitive function. Its probiotic properties are achieved by promoting the proliferation of probiotics and maintaining intestinal barrier function. At the same time, it has anti-adhesion and antibacterial effects, effectively reducing the probability of infection by pathogenic microorganisms. The immunomodulatory function of 6′-sialyllactose enhances the body's defenses by finely regulating the activity of immune cells. Its regulatory effect on intestinal epithelial cells maintains the integrity of the intestinal barrier. Furthermore, 6′-sialyllactose has also shown a positive effect in promoting brain development and improving cognitive function in infants and young children.

[0005] Given its numerous health benefits, the U.S. Food and Drug Administration (FDA) has designated it as a Generally Recognized as Safe (GRAS) substance, and it is widely used in high-end infant formula to mimic the nutritional composition of breast milk and support the healthy development of infants and young children. Future, in-depth interdisciplinary research will further elucidate the molecular mechanisms of 6′-sialyllactose, validate its clinical effects, and evaluate its safety, aiming to optimize its application strategies in areas such as food nutrition and pharmaceutical health.

[0006] In the biosynthesis of human milk oligosaccharides, enzymatic synthesis methods are often limited by the cost of cofactors and substrates, especially the high price of nucleoside sugar substrates, which to a certain extent restricts the economic feasibility of the synthesis process. However, biosynthetic methods, due to their ability to produce 6'-sialyllactose using low-cost carbon sources, show significant economic advantages and production convenience. Despite this, current research focuses on screening and optimizing high-performance sialyltransferases to increase the yield of 6'-sialyllactose. However, the application of α2,6-sialyltransferases in 6'-sialyllactose synthesis has not been fully developed. Finding and developing more efficient α2,6-sialyltransferases remains a key research direction in this field.

[0007] Furthermore, existing research generally employs plasmid-based pathway optimization strategies to achieve the biosynthesis of 6′-sialyllactose. Although introducing plasmids into engineered strains can increase target product yields, plasmid instability and the complexity they introduce into the fermentation process pose significant challenges to the production process. To maintain fermentation stability, the use of antibiotics becomes unavoidable, which not only increases production costs but also raises widespread concerns about potential environmental impacts. Chromosomal integration strategies offer a more precise method for controlling gene dosage and significantly improves the stability of gene expression, thus providing a more sustainable solution for the stable production of 6′-sialyllactose. Chromosomal integration avoids the instability associated with plasmids, reduces reliance on antibiotics, and mitigates environmental risks during production. Therefore, exploring and optimizing chromosomal integration strategies has important theoretical and practical implications for improving the efficiency of 6′-sialyllactose production, reducing costs, and achieving green production. Summary of the Invention

[0008] To address these issues, the present invention screened and identified a novel α2,6-sialyltransferase with the potential to produce 6'-sialyllactose. Genes encoding enzymes in the precursor CMP-Neu5Ac biosynthesis pathway and the α2,6-sialyltransferase were then introduced into Escherichia coli to construct a novel biosynthetic pathway for 6'-sialyllactose, thereby generating an engineered bacterium capable of efficiently producing 6'-sialyllactose. Furthermore, by integrating and expressing the enzymes in the precursor CMP-Neu5Ac biosynthesis pathway and the α2,6-sialyltransferase in the engineered bacterium, the present invention avoids the instability associated with plasmids, reduces reliance on antibiotics during production, and mitigates environmental risks.

[0009] In one aspect, the present application provides the use of a protein in constructing an engineered bacterium that produces 6'-sialyllactose, preparing 6'-sialyllactose, or regulating the production of 6'-sialyllactose by a microorganism, wherein the protein comprises at least any one or more of the following A1)-A9):

[0010] A1) a glycosyltransferase derived from Kingella denitrificans; preferably, the glycosyltransferase sa354 derived from Kingella denitrificans; more preferably, the glycosyltransferase has an amino acid sequence as shown in SEQ ID NO. 8, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to SEQ ID NO. 8;

[0011] A2) a glycosyltransferase from Vespertiliibacter pulmonis; preferably, the glycosyltransferase ed6st from Vespertiliibacter pulmonis; more preferably, the glycosyltransferase has an amino acid sequence as shown in SEQ ID NO. 10, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity thereto;

[0012] A3) a glycosyltransferase derived from Otariodibacter oris; preferably, the glycosyltransferase des31 derived from Otariodibacter oris; more preferably, the glycosyltransferase has an amino acid sequence as shown in SEQ ID NO. 12, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to SEQ ID NO. 12;

[0013] A4) a glycosyltransferase derived from Paramixta manurensis; preferably, the glycosyltransferase pmpd1 derived from Paramixta manurensis; more preferably, the glycosyltransferase has an amino acid sequence as shown in SEQ ID NO. 14, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to SEQ ID NO. 14;

[0014] A5) a glycosyltransferase derived from Pseudoalteromonas rubra; preferably, the glycosyltransferase tw77 derived from Pseudoalteromonas rubra; more preferably, the glycosyltransferase has an amino acid sequence as shown in SEQ ID NO. 16, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to SEQ ID NO. 16;

[0015] A6) a glycosyltransferase from Nicoletella semolina; preferably, the glycosyltransferase ev6st from Nicoletella semolina; more preferably, the glycosyltransferase has an amino acid sequence as shown in SEQ ID NO. 18, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to SEQ ID NO. 18;

[0016] A7) a glycosyltransferase derived from Muribacter muris; preferably, the glycosyltransferase ro6st derived from Muribacter muris; more preferably, the glycosyltransferase has an amino acid sequence as shown in SEQ ID NO. 20, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to SEQ ID NO. 20;

[0017] A8) a glycosyltransferase derived from Simonsiella muelleri; preferably, the glycosyltransferase hmp0133 derived from Simonsiella muelleri; more preferably, the glycosyltransferase has an amino acid sequence as shown in SEQ ID NO. 22, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to SEQ ID NO. 22;

[0018] A9) a glycosyltransferase derived from Pasteurellaceae bacterium; preferably, the glycosyltransferase bmt54 derived from Pasteurelaceae bacterium; more preferably, the glycosyltransferase has an amino acid sequence as shown in SEQ ID NO. 24, or an amino acid sequence having at least 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity thereto.

[0019] More preferably, the glycosyltransferase ev6st from Nicoletella semolina, the glycosyltransferase ro6st from Muribacter muris, the glycosyltransferase hmp0133 from Simonsiella muelleri and / or the glycosyltransferase bmt54 from Pasteurellaceae bacterium.

[0020] This application demonstrates for the first time that the glycosyltransferases sa354, ed6st, des31, pmpd1, tw77, ev6st, ro6st, hmp0133, and bmt54 possess α2,6-sialyltransferase function and can be used as α2,6-sialyltransferases in the production of 6'-sialyllactose. Furthermore, these enzymes possess higher α2,6-sialyltransferase activity than known glycosyltransferases, potentially further increasing 6'-sialyllactose production.

[0021] The new glycosyltransferase screened and obtained in the present application is applied to the process of producing 6'-sialyllactose, and has higher enzyme activity and 6'-sialyllactose yield than known glycosyltransferases.

[0022] Furthermore, the protein includes at least one or more of the following B1)-B11):

[0023] B1) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 8;

[0024] B2) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 10;

[0025] B3) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 12;

[0026] B4) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 14;

[0027] B5) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 16;

[0028] B6) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 18;

[0029] B7) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 20;

[0030] B8) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 22;

[0031] B9) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 24;

[0032] B10) contains a protein having 99% or greater identity with the proteins defined in B1) to B9) and associated with 6'-sialyllactose production;

[0033] B11) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of B1) to B10).

[0034] On the other hand, the present application also provides a biomaterial, which comprises at least one of the following C1)-C3):

[0035] C1) a nucleic acid molecule encoding the protein;

[0036] C2) an expression cassette containing the nucleic acid molecule described in C1);

[0037] C3) A recombinant vector containing the nucleic acid molecule described in C1) and / or a recombinant vector containing the expression cassette described in C2).

[0038] Those skilled in the art will appreciate that the expression cassette described herein may further include functional elements such as a promoter, a terminator, and a marker gene. Those skilled in the art may make routine selections based on actual conditions, as long as the expression of the nucleic acid molecule encoding the protein can be achieved. Excessive restrictions will not be placed on the structure and composition of the expression cassette herein.

[0039] In a preferred embodiment, the promoter is a strong promoter, preferably, the strong promoter is P J23119 .

[0040] A recombinant vector, as described herein, is a vector capable of transporting exogenous DNA or a gene of interest into host cells for amplification and expression. Optionally, the vector may include a nucleic acid encoding the protein, a promoter, and transcriptional and translational stop signals. When preparing a recombinant vector, the nucleic acid encoding the protein may be positioned within the vector so that it is operably linked to appropriate expression control sequences.

[0041] The vector can be a cloning vector or an expression vector. Those skilled in the art can select a vector according to the actual situation, including but not limited to: a plasmid, a phage (such as lambda phage or M13 filamentous phage), a cosmid (i.e., a cosmid), or a viral vector, without further limitation. In a preferred embodiment, the vector is pETDuet-1 and pCDFDuet-1.

[0042] It is understandable that those skilled in the art can select appropriate gene editing systems and gene editing methods according to actual conditions to complete the transformation of the above-mentioned mutants.

[0043] Furthermore, the nucleic acid molecule described in C1) includes any one or more of D1) to D10):

[0044] D1) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 9;

[0045] D2) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 11;

[0046] D3) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 13;

[0047] D4) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 15;

[0048] D5) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 17;

[0049] D6) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 19;

[0050] D7) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 21;

[0051] D8) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 23;

[0052] D9) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 25;

[0053] D10) A nucleic acid molecule having 80% or greater identity with the nucleic acid molecule described in any one of D1) to D9).

[0054] Among them, D10) is a nucleic acid molecule that has 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%, or 99.9% or more identity with the nucleic acid molecule described in any one of D1) to D9).

[0055] Preferably,

[0056] The amino acid sequence of ev6st is shown in SEQ ID NO. 18, and the nucleotide sequence is shown in SEQ ID NO. 19;

[0057] The amino acid sequence of ro6st is shown in SEQ ID NO.20, and the nucleotide sequence is shown in SEQ ID NO.21;

[0058] The amino acid sequence of hmp0133 is shown in SEQ ID NO. 22, and the nucleotide sequence is shown in SEQ ID NO. 23;

[0059] The amino acid sequence of bmt54 is shown in SEQ ID NO. 24, and the nucleotide sequence is shown in SEQ ID NO. 25.

[0060] On the other hand, the present application also provides a recombinant microorganism, which comprises any one or more of the following E1)-E4):

[0061] E1) a recombinant microorganism containing the protein;

[0062] E2) a recombinant microorganism containing the nucleic acid molecule;

[0063] E3) a recombinant microorganism containing the expression cassette;

[0064] E4) A recombinant microorganism containing the recombinant vector.

[0065] Furthermore, the recombinant microorganism further contains a gene for expressing a precursor enzyme;

[0066] Preferably, the precursor enzyme is a precursor CMP-Neu5Ac synthesis pathway enzyme;

[0067] Preferably, the precursor enzyme is neuBCA derived from Campylobacter jejuni.

[0068] Preferably, the amino acid sequence of the neuBCA is shown as SEQ ID NO.26, and the nucleotide sequence is shown as SEQ ID NO.27.

[0069] The neuBCA is composed of neuB, neuC, and neuA connected in series.

[0070] Preferably, the gene expressing the precursor enzyme and the nucleic acid molecule encoding the glycosyltransferase can be inserted into the genome of the recombinant microorganism for integrated expression or exist on a plasmid in a free state, as long as expression can be achieved.

[0071] More preferably, the gene expressing the precursor enzyme and the nucleic acid molecule encoding the protein are inserted into the genome of the recombinant microorganism for integrated expression.

[0072] More preferably, the integration site of the precursor enzyme includes manXYZ and / or pheA.

[0073] More preferably, the integration site of the precursor enzyme includes manXYZ, arsB and / or curA.

[0074] More preferably, the integration site of the precursor enzyme includes manXYZ, arsB, curA and / or pheA.

[0075] In a preferred embodiment, the integration site of the precursor enzyme includes manXYZ and pheA.

[0076] In a preferred embodiment, the integration site of the precursor enzyme includes manXYZ, arsB, and curA.

[0077] More preferably, the integration site for the glycosyltransferase includes poxB.

[0078] More preferably, the integration site of the glycosyltransferase includes poxB, yghWX and / or ldhA.

[0079] In a preferred embodiment, the integration site for the glycosyltransferase comprises poxB.

[0080] In a preferred embodiment, the integration sites of the glycosyltransferase include poxB, yghWX, and ldhA.

[0081] Furthermore, the expression ratio of the precursor enzyme to the protein in the recombinant microorganism is (1-3):1.

[0082] In a preferred embodiment, the expression ratio of the precursor enzyme to the protein in the recombinant microorganism may be 1:1, 1.5:1, 2:1, 2.5:1, or 3:1.

[0083] Furthermore, the expression ratio of the precursor enzyme to the protein in the recombinant microorganism is (1-2):1.

[0084] Preferably, the expression ratio of the precursor enzyme to the protein in the recombinant microorganism is any one of 2:1, 1:1, and 1.5:1.

[0085] The present application provides a more precise gene dosage control method by integrating and expressing the target gene on the chromosome of the engineered bacteria, which can significantly improve the stability of gene expression.

[0086] Furthermore, the recombinant microorganism also expresses glmM, which encodes glucosamine-6-phosphate dehydratase, to convert glucosamine-6-phosphate (GlcN-6-P) into glucosamine-1-phosphate (GlcN-1-P); glmU, which encodes a bifunctional enzyme having both glucosamine-1-phosphate uridyltransferase and acetyltransferase activities, to catalyze the synthesis of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) from glucosamine-1-phosphate (GlcN-1-P), acetyl coenzyme A (CoA) and uridine triphosphate (UTP); and glmS, which encodes glucosamine-6-phosphate synthase, to catalyze the hydrolysis of glutamine to produce ammonia, which then reacts with fructose-6-phosphate (Fru-6-P) to produce glucosamine-6-phosphate (GlcN-6-P).

[0087] in,

[0088] The amino acid sequence of glmM is shown in SEQ ID NO. 29, and the nucleotide sequence is shown in SEQ ID NO. 30;

[0089] The amino acid sequence of glmS is shown in SEQ ID NO. 31, and the nucleotide sequence is shown in SEQ ID NO. 32;

[0090] The amino acid sequence of glmU is shown in SEQ ID NO.33, and the nucleotide sequence is shown in SEQ ID NO.34.

[0091] In a preferred embodiment, the gene expression level can be precisely controlled by controlling the number of integrated genes.

[0092] Furthermore, the recombinant microorganism is any one of the following F1)-F2):

[0093] F1) Escherichia bacteria;

[0094] F2) Escherichia coli.

[0095] Preferably, Escherichia coli.

[0096] In a preferred embodiment, the Escherichia coli is Escherichia coli BL21 (DE3) or Escherichia coli MG1655.

[0097] More preferably, the recombinant microorganism is further subjected to any one or more of the following modifications G1) to G4):

[0098] G1) silencing and / or weakening the expression of the β-galactosidase gene; preferably, the β-galactosidase gene is lacZ;

[0099] G2) silencing and / or weakening the expression of the N-acetylneuraminic acid aldolase gene; preferably, the N-acetylneuraminic acid aldolase gene is nanA;

[0100] G3) silencing and / or weakening the expression of the N-acetylmannosamine kinase gene; preferably, the N-acetylmannosamine kinase gene is nanK;

[0101] G4) Silencing and / or weakening the expression of the UDP-glucose lipid carrier transferase gene; preferably, the UDP-glucose lipid carrier transferase gene is wcaJ.

[0102] In a preferred embodiment, the silencing and / or weakening is achieved by gene knockout. It will be appreciated by those skilled in the art that other methods can be used to achieve silencing and / or weakening of the target gene in actual operation, as long as the effect of reducing the expression of the target gene can be achieved.

[0103] In a preferred embodiment, lacZ, nanA and nanK in Escherichia coli BL21 (DE3) are knocked out, ev6st is integrated at the poxB site, and neuBCA is integrated at the manXYZ site to obtain the recombinant microorganism.

[0104] In a preferred embodiment, lacZ, nanA and nanK in Escherichia coli BL21 (DE3) are knocked out, ev6st is integrated at the poxB site, and neuBCA is integrated at the manXYZ and pheA sites to obtain the recombinant microorganism.

[0105] In a preferred embodiment, wcaJ, lacZ, nanA and nanK in Escherichia coli MG1655 are knocked out, ev6st is integrated at the poxB, yghWX and / or ldhA sites, neuBCA is integrated at the manXYZ, arsB and / or curA sites, glmM is integrated at the adhE site, and glmS is integrated at the xylB site to obtain the recombinant microorganism.

[0106] In a preferred embodiment, lacZ, nanA and nanK are knocked out in E. coli MG1655, Hmp0133 is integrated at the poxB, yghWX and / or ldhA sites, neuBCA is integrated at the manXYZ and arsB sites, glmM is integrated at the adhE site, P is knocked out lacY The promoter was replaced by P J23119 Promoter, to obtain the recombinant microorganism.

[0107] In a preferred embodiment, lacZ, nanA and nanK are knocked out in E. coli MG1655, Hmp0133 is integrated at the poxB, yghWX and / or ldhA sites, neuBCA is integrated at the manXYZ, arsB and / or curA sites, glmM is integrated at the adhE site, glmS is integrated at the xylB site, P is knocked out lacY The promoter was replaced by P J23119 Promoter, to obtain the recombinant microorganism.

[0108] In a preferred embodiment, lacZ, nanA, and nanK are knocked out in E. coli MG1655, Hmp0133 is integrated at the poxB, yghWX, and / or ldhA sites, neuBCA is integrated at the manXYZ, arsB, and / or curA sites, glmM is integrated at the adhE site, glmS is integrated at the xylB site, glmU is integrated at the fuci site, P is knocked out lacY The promoter was replaced by P J23119 Promoter, to obtain the recombinant microorganism.

[0109] On the other hand, the present application also provides a whole-cell catalyst comprising the recombinant microorganism.

[0110] On the other hand, the present application also provides a method for preparing 6'-sialyllactose, which comprises: using the recombinant microorganism or the whole-cell catalyst to ferment and prepare 6'-sialyllactose.

[0111] Preferably, the fermentation process does not contain antibiotics.

[0112] Among them, 6'-sialyllactose can be prepared by fermentation using a general method.

[0113] In a preferred embodiment, the method comprises:

[0114] The recombinant microorganism or the whole-cell catalyst is inoculated into the culture medium and cultured at 25°C to 40°C, 100-200 rpm until the OD 600 The concentration of the iodine was 0.6-0.8, lactose and IPTG were added, and the culture was induced at 25-40°C and 100-200 rpm for 48-72 hours.

[0115] In a preferred embodiment, the method comprises:

[0116] Step 1: inoculating the recombinant microorganism or the whole-cell catalyst into a culture medium and culturing for 12 to 24 hours to obtain a first-level seed solution;

[0117] Step 2: inoculate the primary seed solution into the culture medium and perform the first expansion culture at 25°C to 40°C and 100-200 rpm;

[0118] Step 3: Cultivate to OD 600 =1.5~2, then transferred to fermenter and cultured at 37℃. 600 When the temperature reaches 30-33°C, cool to 25°C, add lactose and IPTG, and feed the fermentation solution to ensure that the final concentration of glycerol is maintained at 2-8 g / L and the final concentration of lactose is maintained at 2-5 g / L. The pH is maintained at 6.8±0.1 throughout the process, and the dissolved oxygen is controlled at 20%-30% by adjusting the stirring speed and ventilation volume.

[0119] On the other hand, the present application also provides the use of the biomaterial, the recombinant microorganism, or the whole-cell catalyst in constructing 6'-sialyllactose-producing bacteria, preparing 6'-sialyllactose, or regulating the production of 6'-sialyllactose by microorganisms.

[0120] The present invention has the following beneficial effects:

[0121] The present invention first introduces genes encoding enzymes of the precursor CMP-Neu5Ac synthesis pathway and α2,6-sialyltransferase into Escherichia coli EZAK (Escherichia coli BL21 (DE3) knockout of the competing pathway genes β-galactosidase gene lacZ, N-acetylneuraminic acid aldolase gene nanA, and N-acetylmannosamine kinase gene nanK) through two plasmids, thereby constructing a new biosynthetic pathway for 6′-sialyllactose. Using this dual-plasmid system, the present invention preliminarily screened out potential α2,6-sialyltransferases (glycosyltransferase ev6st from Nicoletella semolina, glycosyltransferase ro6st from Muribacter muris, α2,3-sialyltransferase hmp0133 from Simonsiella muelleri, and α2,3-sialyltransferase bmt54 from Pasteurellaceaebacterium) from four reported α2,6-sialyltransferases and nine novel α2,6-sialyltransferases. This provides new α2,6-sialyltransferases and new biomaterials for constructing engineered bacteria that produce 6'-sialyllactose.

[0122] In addition, in the present invention, a strong promoter (P J23119) under the control of the gene cluster neuBCA, which synthesizes CMP-Neu5Ac, and the integration of the α2,6-sialyltransferase gene ev6st or Hmp0133 at the poxB, yghWX, and ldhA sites to generate high-yield 6'-sialyllactose strains. This efficient integration and expression of the α2,6-sialyltransferase in engineered 6'-sialyllactose-producing bacteria avoids plasmid-related instability, reduces reliance on antibiotics during production, and mitigates environmental risks. This provides a new approach for the construction of engineered 6'-sialyllactose-producing bacteria that balances safety and production efficiency.

[0123] In batch culture experiments, the engineered strain HXM-20 achieved an extracellular yield of 5.68 g / L of 6′-sialyllactose in shake flasks, the highest yield achieved to date. In 5L fermentation experiments, the engineered strain HM23MS achieved an extracellular yield of 17.22 g / L of 6′-sialyllactose, achieving stable fermentation under plasmid-free conditions and demonstrating significant production potential for large-scale industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] 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:

[0125] Figure 1 Figure A is a schematic diagram of the construction of the pET-BCA plasmid (left) and the pCD-α2,6-sialyltransferase plasmid (right), and Figure B is a comparison of the 6′-sialyllactose yields of various exogenous α2,6-sialyltransferases EZAK.

[0126] Figure 2 LC-MS images of 6′-sialyllactose standard product and HXM-15, wherein A is the liquid phase image of HXM-15, B is the mass spectrum of HXM-15, C is the liquid phase image of 6′-sialyllactose standard product, and D is the mass spectrum of 6′-sialyllactose standard product;

[0127] Figure 3 The figure shows the fermentation yield of 6′-sialyllactose in a 5L fermenter of HXM-20;

[0128] Figure 4 The figure shows the fermentation yield of 6′-sialyllactose in a 5 L fermenter of EM33MS;

[0129] Figure 5 This is a graph showing the fermentation yield of 6′-sialyllactose in a 5L fermenter of HM23MS. DETAILED DESCRIPTION

[0130] Technical terms:

[0131] Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution research.

[0132] Recombination: In a broad sense, any gene exchange process that causes genotype changes is called recombination.

[0133] Expression cassette: An expression cassette is a set of DNA sequences consisting of a promoter, target gene, and reporter gene that can be expressed in specific tissues and easily detected.

[0134] Recombinant vector: A recombinant vector is a vector that transfers the target gene into the basic skeleton of a cloning vector, thereby enabling the target gene to be expressed.

[0135] Recombinant microorganisms (engineered bacteria): Fungal cell lines that use genetic engineering methods to efficiently express exogenous genes.

[0136] 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 participating in the catalytic process is the whole-cell catalyst.

[0137] Episomal expression: Episomal expression is the expression of target genes using episomal plasmids. Episomal plasmids are independent DNA molecules that exist in cells and have the ability to replicate and transmit independently. They are widely used in genetic engineering and molecular biology research.

[0138] Integrative expression: expression that occurs when a gene is integrated into the genome.

[0139] In order to more clearly illustrate the overall concept of the present application, the following is a detailed description of the embodiments in conjunction with the accompanying drawings. 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.

[0140] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0141] The plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples were commercially available products, and specific procedures were performed according to the kit instructions. Competent E. coli cells were prepared using the TAKARA kit, and specific procedures were performed according to the kit instructions. Conventional procedures such as colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation, competent cell preparation, and bacterial genome extraction and storage were performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0142] The tandem target gene neuBCA involved in the following examples was constructed by Suzhou Anshengda Company on the vector of pETDuet-1 plasmid MCS1 multiple cloning site to generate plasmid pET-BCA; the target genes st6119, pdst1, st6224, hac1268, sa354, ed6st, des31, pmpd1, tw77, ev6st, ro6st, hmp0133 and bmt54 were constructed by Suzhou Anshengda Company on the expression vector pC Between the multiple cloning site 1 NcoI site and the NotI site of DFDuet-1, plasmids pCD-St6119, pCD-PdST1, pCD-St6224, pCD-HAC1268, pCD-SA354, pCD-ED6ST, pCD-DES31, pCD-PMPD1, pCD-TW77, pCD-EV6ST, pCD-RO6ST, pCD-HMP0133, and pCD-BMT54 were generated, respectively.

[0143] in,

[0144] The amino acid sequence of neuBCA is shown in SEQ ID NO. 26, and the nucleotide sequence is shown in SEQ ID NO. 27;

[0145] The NCBI accession number of neuB is AAK91726.1, and the nucleotide sequence is shown in SEQ ID NO. 1;

[0146] The NCBI accession number of neuC is AAK91727.1, and the nucleotide sequence is shown in SEQ ID NO. 2;

[0147] The NCBI accession number of neuA is AAK91728.1, and the nucleotide sequence is shown in SEQ ID NO. 3;

[0148] The NCBI accession number of st6119 is BAI49484, and the nucleotide sequence is shown in SEQ ID NO. 4;

[0149] The NCBI accession number of pdst1 is BAA25316, and the nucleotide sequence is shown in SEQ ID NO. 5;

[0150] The NCBI accession number of st6224 is BAF92026, and the nucleotide sequence is shown in SEQ ID NO. 6;

[0151] The NCBI accession number of hac1268 is NC_008229.1, and the nucleotide sequence is shown in SEQ ID NO. 7;

[0152] The NCBI accession number of sa354 is WP_003782123, the amino acid sequence is shown in SEQ ID NO. 8, and the nucleotide sequence is shown in SEQ ID NO. 9;

[0153] The NCBI accession number of ed6st is WP_124210845, the amino acid sequence is shown in SEQ ID NO. 10, and the nucleotide sequence is shown in SEQ ID NO. 11;

[0154] The NCBI accession number of des31 is WP_121120834.1, the amino acid sequence is shown in SEQ ID NO. 12, and the nucleotide sequence is shown in SEQ ID NO. 13;

[0155] The NCBI accession number of pmpd1 is QKJ87768.1, the amino acid sequence is shown in SEQ ID NO. 14, and the nucleotide sequence is shown in SEQ ID NO. 15;

[0156] The NCBI accession number of tw77 is WP_046004788.1, the amino acid sequence is shown in SEQ ID NO. 16, and the nucleotide sequence is shown in SEQ ID NO. 17;

[0157] The NCBI accession number of ev6st is WP_132500470, the amino acid sequence is shown in SEQ ID NO. 18, and the nucleotide sequence is shown in SEQ ID NO. 19;

[0158] The NCBI accession number of ro6st is WP_047977564, the amino acid sequence is shown in SEQ ID NO. 20, and the nucleotide sequence is shown in SEQ ID NO. 21;

[0159] The NCBI accession number of hmp0133 is AUX62520.1, the amino acid sequence is shown in SEQ ID NO. 22, and the nucleotide sequence is shown in SEQ ID NO. 23;

[0160] The NCBI accession number of bmt54 is OOH88723.1, the amino acid sequence is shown in SEQ ID NO. 24, and the nucleotide sequence is shown in SEQ ID NO. 25.

[0161] In the following examples, the CRISPR / Cas9 dual-plasmid gene editing system has been disclosed in the literature: Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., Yang, S. Multigene Editing in the Escherichia Coli Genome via the CRISPR / Cas9 System[J]. Applied and Environmental Microbiology, 2015, 81(7), 2506-2514.

[0162] The following relates to the recombinant E. coli EZAK, which is disclosed in Chinese Patent Publication No. CN114874966A. Specifically, the construction method of the recombinant EZAK includes: knocking out the β-galactosidase gene lacZ, the N-acetylneuraminic acid aldolase gene nanA, and the N-acetylmannosamine kinase gene nanK in E. coli BL21 (DE3).

[0163] The culture medium and reagents involved in the following examples:

[0164] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and the balance is water.

[0165] LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder, and the balance is water.

[0166] Define dmedium (DM) fermentation medium: 20 g / L glycerol, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate, 5 g / L yeast extract, and 10 ml / L trace metal elements (the balance is water). Trace metal elements include: 10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L anhydrous copper sulfate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, 2.0 g / L calcium chloride dihydrate, and the balance is water. Adjust the pH to 6.8 with sodium hydroxide and sterilize at 115°C for 30 min. Add 4.5 mg / L thiamine before use.

[0167] Antibiotic concentration: ampicillin 100 mg / L (liquid culture medium), streptomycin 50 mg / L.

[0168] Fermentation feed solution: 600 g / L glycerol, 0.2 g / L thiamine, 200 g / L lactose, 50% ammonia water (w / v), and the balance water.

[0169] HPLC detection conditions: 1 mL of fermentation broth was centrifuged at 12,000 rpm for 5 min, and the supernatant was collected for HPLC determination.

[0170] (1) Lactose and glycerol detection: by high performance liquid chromatography (HPLC) system (Agilent 1260 Infinity II); chromatographic column: Rezex ROA-organic acid H + (8%); detector: Agilent 1260RID differential detector; mobile phase: 0.5 mM H2SO4; flow rate: 0.6 mL / min; column temperature: 60°C; injection volume: 10 μL.

[0171] (2) Detection of 6′-sialyllactose: by high performance liquid chromatography (HPLC) system (Agilent 1260 Infinity II); chromatographic column: Zorbax NH2; detector: Agilent 1260 RID differential detector; mobile phase: 50% acetonitrile, 2 mM MgCl2, 10 mM H3PO4; flow rate: 0.8 mL / min; column temperature: 30°C; injection volume: 10 μL.

[0172] 6′-sialyllactose shake flask fermentation process:

[0173] The constructed strain was inoculated into LB liquid medium containing corresponding resistance or no resistance, and cultured overnight for 12 h to obtain seed liquid. The seed liquid was inoculated into DM fermentation medium and cultured at 37 ° C, 200 rpm until OD 600 The pH value was 0.6-0.8, and lactose and 0.5 mM IPTG were added at a final concentration of 3 g / L. The induction culture was continued at 25°C and 200 rpm for 72 h.

[0174] 6′-sialyllactose fed-batch fermentation process:

[0175] A single colony of recombinant E. coli was picked from the plate and inoculated into LB medium without resistance for 12 h as the first-level seed liquid; the first-level seed liquid was inoculated into 150 mL of DM fermentation medium and cultured at 37°C and 200 rpm until the OD 600 =1.5 and then transferred to a 5L fermentor containing 2L DM fermentation medium. The initial temperature was maintained at 37°C. 600When the temperature reaches 30-33°C, cool to 25°C and add lactose to a final concentration of 2 g / L and 0.2 mM IPTG. Fermentation feed solution is then fed to maintain a final concentration of glycerol at 2-8 g / L and lactose at 2-5 g / L. The pH is maintained at 6.8 ± 0.1 throughout the process, and defoamer is added to control foaming. Dissolved oxygen is maintained at 20%-30% by adjusting the stirring speed and aeration rate.

[0176] Example 1 Construction of recombinant expression vector

[0177] The specific steps for constructing the recombinant expression vector used to transform Escherichia coli are as follows:

[0178] (1) Obtaining the neuBCA gene fragment and constructing the plasmid pET-BCA:

[0179] The codon-optimized Campylobacter jejuni-derived gene neuBCA (neuBCA is composed of neuB, neuC, and neuA in series) was constructed between the NcoI and BamHI sites of the MCS1 multiple cloning site of the expression vector pETDuet-1 to form the recombinant plasmid pET-BCA (such as Figure 1 A left).

[0180] (2) Obtaining the α2,6-sialyltransferase gene fragment and constructing the plasmid pCD-α2,6-sialyltransferase:

[0181] Using NST (NCBI accession number AAC44541) as a template, blast was performed on the Uniprot website (https: / / www.uniprot.org / ), and the following 13 new α2,6-sialyltransferase genes were obtained: st6119, pdst1, st6224, hac1268, sa354, ed6st, des31, pmpd1, tw77, ev6st, ro6st, hmp0133, and bmt54. Taking the construction of pCD-St6119 plasmid as an example, the st6119 fragment was PCR-generated using the synthetic st6119 gene as a template using primers St6119-PF and St6119-PR, and the pCD vector fragment was PCR-generated using primers pCD-VF and pCD-VR using the pCDFDuet-1 plasmid as a template. The fragment and the vector were ligated by Gibson assembly and transformed into E. coli JM109 to obtain recombinant pCD-St6119. pCD-PdST1, pCD-St6224, pCD-HAC1268, pCD-SA354, pCD-ED6ST, pCD-DES31, pCD-PMPD1, pCD-TW77, pCD-EV6ST, pCD-RO6ST, pCD-HMP0133, and pCD-BMT54 (e.g. Figure 1 The plasmid construction of A (right) is similar.

[0182] The primers involved are shown in Table 1.

[0183] Table 1

[0184]

[0185] Example 2 Fermentation production of 6'-sialyllactose by recombinant strains of different sialyltransferases

[0186] The genes lacZ, nanA and nanK in Escherichia coli BL21 (DE3) were knocked out. The specific steps refer to the patent publication number CN114874966A to obtain the recombinant bacterium EZAK.

[0187] (1) Production of 6'-sialyllactose by shake flask fermentation

[0188] The pET-BCA constructed in Example 1 and pCD-St6119, pCD-PdST1, pCD-St6224, pCD-HAC1268, pCD-SA354, pCD-ED6ST, pCD-DES31, pCD-PMPD1, pCD-TW77, pCD-EV6ST, pCD-RO6ST, pCD-HMP0133, and pCD-BMT54 were respectively transformed into the above-constructed host EZAK to obtain recombinant strains HXM-06, HXM-07, HXM-08, HXM-09, HXM-10, HXM-11, HXM-12, HXM-13, HXM-14, HXM-15, HXM-16, HXM-17, and HXM-18.

[0189] The recombinant strains were inoculated into LB liquid medium containing the corresponding antibiotics, cultured at 37°C, 200 rpm, and cultured overnight for 12 h to obtain seed liquid. The seed liquid was inoculated with 2% of the inoculum into 25 mL of DM fermentation medium and cultured at 37°C, 200 rpm until the OD 600 When the pH value was 0.6-0.8, 3 g / L lactose and 0.5 mM IPTG were added for induction and cultured at 25°C and 200 rpm for 72 h. 1 mL of fermentation broth was taken and the supernatant was collected. The 6′-sialyllactose content in the supernatant was determined by HPLC. The results are shown in Table 2. Figure 1 B.

[0190] (2) Screening of α2,6-sialyltransferase

[0191] As shown in Table 2 and Figure 1Results B show that most of the 13 α2,6-sialyltransferases (St6119, PdST1, St6224, HAC1268, SA354, ED6ST, DES31, PMPD1, TW77, EV6ST, RO6ST, HMP0133, and BMT54) showed good fermentation potential. Among them, the 6'-sialyllactose production of strain HXM-16 (containing RO6ST enzyme) was 2.62 g / L, the 6'-sialyllactose production of strain HXM-15 (containing Ev6ST enzyme) was 2.94 g / L, the 6'-sialyllactose production of strain HXM-17 (containing HMP0133 enzyme) was 3.26 g / L, and the 6'-sialyllactose production of strain HXM18 (containing BMT54 enzyme) was 2.31 g / L, exceeding the previously reported The yields of the four enzymes of Dao (strain HXM-06 containing St6119 enzyme produced 1.59 g / L of 6'-sialyllactose, strain HXM-07 containing PdST1 enzyme produced 1.89 g / L of 6'-sialyllactose, strain HXM-08 containing St6224 enzyme produced 1.64 g / L of 6'-sialyllactose, and strain HXM-09 containing HAC1268 enzyme produced 2.17 g / L of 6'-sialyllactose) were measured.

[0192] In addition, the LC-MS analysis of the shake flask culture supernatant of HXM-15 was performed, and the results were as follows: Figure 2 As shown, the m / z values ​​of the HXM-15 sample and the 6'-sialyllactose standard are both 632, which confirms that the product generated by HXM-15 is 6'-sialyllactose.

[0193] Table 2

[0194]

[0195] Example 3 Construction of a high-yield plasmid-free strain by gene integration

[0196] We attempted to integrate the neuBCA and ev6st genes into EZAK to replace the use of plasmids. We further introduced the constitutive promoter P J23119 To replace the original promoter, the expression levels of neuBCA and ev6st were increased, thereby obtaining the recombinant bacteria HXM-19.

[0197] The specific steps are as follows (the primer sequences involved are shown in Table 3):

[0198] (1) Using EZAK as the initial bacteria, manXYZ-UP-F / R and manXYZ-DH-F / R were used to amplify the upstream and downstream fragments of the manXYZ site by PCR. Then, using the neuBCA gene cluster as a template, neuB-J23-F and neuA-T7-R were used to amplify the fragment containing a strong promoter (P J23119 The three fragments were connected by PCR to obtain the donor DNA fragment.

[0199] (2) Using the original pTargetF plasmid as a template and manXYZ-N20-F / R as primers, PCR amplification was performed to replace the N20 sequence on the original plasmid with an N20 sequence complementary to the poxB sequence, resulting in the pTargetF plasmid pTargetF-manXYZ with a targeted manXYZ site. The PCR product was treated with Dpn I enzyme to remove the template DNA, transformed into competent Escherichia coli JM109, plated on LB plates (containing spectinomycin), and cultured at 37°C to extract the plasmid and sequence it.

[0200] (3) Take the pCas plasmid and E. coli EZAK competent cells, place them on ice until the competent cells thaw, add the plasmid to the competent cells, and mix gently. Ice bath for 20 minutes, heat shock at 42°C for 90 seconds, and immediately place on ice. Add 1 mL of LB medium and culture at 30°C, 200 rpm for 1 hour. Take the concentrated bacterial solution, evenly spread it on an LB plate (containing kanamycin), and culture it upside down at 30°C overnight to become E. coli EZAK / pCas.

[0201] (4) Pick a single colony of E. coli EZAK / pCas and place it in LB medium, culture it at 30℃ for 1 hour, and add L-arabinose with a final concentration of 30mM to induce the expression of the pCas-λ-red system. 600 When the concentration reaches 0.6-0.8, prepare the E. coli EZAK / pCas electroporation competent cells.

[0202] (5) 200 ng of pTargetF-manXYZ plasmid and 800 ng of donor DNA fragment were electroporated into E. coli EZAK / pCas electroporation competent cells, spread on LB plates (kanamycin and spectinomycin), cultured at 30°C for 24 h, and PCR colony verification was performed.

[0203] (6) The positive clones were transferred to LB tubes, and IPTG and 30 mg / L kanamycin were added to a final concentration of 1 mM. The cells were cultured at 30°C for 12 h to remove the pTargetF-manXYZ plasmid. The pCas plasmid was removed after culturing at 42°C for 12 h. The recombinant strain EZAKB was obtained.

[0204] (7) Similarly, using EZAKB as the initial strain, poxB-UP-F / R and poxB-DH-F / R were used to amplify the upstream and downstream fragments of the poxB site by PCR. Then, using the ev6st gene as a template, EV6ST-J23-F and EV6ST-T7-R were used to amplify the fragment containing the strong promoter (P J23119 The three fragments were connected by PCR to obtain the donor DNA fragment.

[0205] (8) Using the original pTargetF plasmid as a template and poxB-N20-F / R as primers, PCR amplification was performed to replace the N20 sequence on the original plasmid with an N20 sequence complementary to the poxB sequence, resulting in the pTargetF plasmid pTargetF-poxB with a targeted poxB site. The PCR product was treated with Dpn I enzyme to remove the template DNA, transformed into competent Escherichia coli JM109, plated on LB plates (containing spectinomycin), and cultured at 37°C to extract the plasmid and sequence it.

[0206] (9) Take the pCas plasmid and E. coli EZAKB competent cells, place them on ice until the competent cells thaw, add the plasmid to the competent cells, and mix gently. Ice bath for 20 minutes, heat shock at 42°C for 90 seconds, and immediately place on ice. Add 1 mL of LB medium and culture at 30°C, 200 rpm for 1 hour. Take the concentrated bacterial solution, evenly spread it on an LB plate (containing kanamycin), and culture it upside down at 30°C overnight to become E. coli EZAKB / pCas.

[0207] (10) A single colony of E. coli EZAKB / pCas was picked and placed in LB medium, cultured at 30°C for 1 h, and L-arabinose was added at a final concentration of 30 mM to induce the expression of the pCas-λ-red system. 600 When the concentration reaches 0.6-0.8, prepare the E. coli EZAKB / pCas electroporation competent cells.

[0208] (11) 200 ng of pTargetF-poxB plasmid and 800 ng of donor DNA fragment were electroporated into E. coli EZAKB / pCas electroporation competent cells, spread on LB plates (kanamycin and spectinomycin), cultured at 30 °C for 24 h, and PCR colony verification was performed.

[0209] (12) The positive clones were transferred to LB tubes, and IPTG and 30 mg / L kanamycin were added to a final concentration of 1 mM. The cells were cultured at 30°C for 12 h to remove the pTargetF-poxB plasmid. The pCas plasmid was removed after culturing at 42°C for 12 h. The recombinant strain HXM-19 was finally obtained.

[0210] The primers involved are shown in Table 3. The fermentation process and the determination of 6′-sialyllactose production were the same as those in Example 2.

[0211] The results are shown in Table 4. Under plasmid-free conditions, the 6'-sialyllactose production of HXM-19 reached 2.47 g / L, proving that expressing both neuBCA and ev6st genes in E. coli as the host bacteria can significantly increase the production of 6'-sialyllactose.

[0212] Table 3

[0213]

[0214]

[0215] Table 4

[0216]

[0217] Example 4 Precise gene dosage control of key enzyme genes through chromosomal integration

[0218] Based on HXM-19, neuBCA was integrated at the pheA site to construct HXM-20. Among them, the ratio of the precursor enzyme gene neuBCA to the α2,6-sialyltransferase gene ev6st in HXM-19 is 1:1, and the ratio of the precursor enzyme gene neuBCA to the α2,6-sialyltransferase gene ev6st in HXM-20 is 2:1. The specific steps of gene integration are the same as those in Example 3. The fermentation process and detection method are the same as those in Example 2. The primers involved are shown in Table 5, and the constructed strains and fermentation results are shown in Table 6. HXM-20 produced 5.68 g / L of 6'-sialyllactose, which is 2.3 times the yield of HXM-19 and 93.2% higher than that of HXM-15. It is the highest yield achieved in shake flask fermentation so far.

[0219] Table 5

[0220]

[0221] Table 6

[0222]

[0223] Example 5: Efficient production of 6'-sialyllactose in an engineered bacterial fermenter

[0224] In order to further verify the effectiveness of the 6'-sialyllactose synthesis method and improve the yield of 6'-sialyllactose, the engineered bacteria HXM-20 seed solution was inoculated into a 2L DM fermentation medium at a 7.5% (v / v) inoculation rate, and the initial temperature was maintained at 37°C. When OD 600 When the temperature reaches 30-33, cool it down to 25℃, and add lactose with a final concentration of 2g / L and IPTG with a final concentration of 0.2mM to induce expression. Subsequently, the fermentation feed solution is added to ensure that the final concentration of glycerol is maintained at 2-8g / L, the final concentration of lactose is maintained at 2-5g / L, the pH is maintained at 6.8±0.1 throughout the process, and the foam is controlled by adding defoaming agent. The dissolved oxygen is controlled at 20%-30% by adjusting the stirring speed and ventilation volume. After the entire culture process reaches 67h, the bacterial OD 600 The extracellular production of 74,6'-sialyllactose reached the highest level, reaching 15.35g / L (e.g. Figure 3 ).

[0225] Example 6: Construction of a plasmid-free strain with high 6'-sialyllactose production based on EV6ST enzyme in K12-MG1655 host strain

[0226] Using E. coli MG1655 as the chassis, the UDP-glucose lipid carrier transferase gene wcaJ and the β-galactosidase gene lacZ were knocked out on the genome by gene editing technology CRISPR / Cas9, and the original promoter of lactose permease lacY (P lacY The nucleotide sequence is shown in SEQ ID NO.35) and replaced with the constitutive promoter P J23119 , obtain the MGC02 genetically engineered bacteria (for specific steps, refer to patent publication number CN117305211A).

[0227] The MAK engineered strain (MG1655ΔwcaJΔlacZΔP) was obtained by knocking out the N-acetylneuraminic acid aldolase gene nanA and the N-acetylmannosamine kinase gene nanK on the MGC02 host. lacY ::P J23119 ΔnanAΔnanK).

[0228] Using MAK as the starting strain, the production of 6'-sialyllactose was significantly improved by introducing different copies of the exogenous tandem gene neuBCA, exogenous α2,6-sialyltransferase, and enhanced precursor genes.

[0229] The specific strain transformation method is described in Example 3. One or more copies of the constitutive promoter P are integrated into the manXYZ, arsB, and curA sites of the MAK host genome. J23119Under the control of neuBCA, one or more copies of the constitutive promoter P are integrated at the poxB, yghWX, and ldhA sites. J23119 The engineered bacteria EM11, EM12, EM21, EM22, EM32 and EM33 were obtained by expressing the α2,6-sialyltransferase Ev6ST under its control.

[0230] A copy of the constitutive promoter P was integrated into the adhE site of the engineered bacterium EM33 genome. J23119 The engineered bacteria EM33M were obtained by controlling glmM.

[0231] A copy of the constitutive promoter P was integrated into the xylB site of the engineered strain EM33M genome. J23119 The engineered bacteria EM33MS were obtained by expressing glmS under its control.

[0232] in,

[0233] The amino acid sequence of glmM is shown in SEQ ID NO. 29, and the nucleotide sequence is shown in SEQ ID NO. 30;

[0234] The amino acid sequence of glmS is shown in SEQ ID NO. 31, and the nucleotide sequence is shown in SEQ ID NO. 32;

[0235] The primers involved in the above method are shown in Table 7, the engineering bacteria information is shown in Table 8, and the rest is the same as Example 3.

[0236] Production of 6'-sialyllactose by shake flask fermentation:

[0237] Each of the nine engineered strains constructed above was inoculated into LB liquid medium and cultured overnight at 37°C, 200 rpm, for 12 hours to obtain seed liquid. A 2% portion of the seed liquid was inoculated into 25 mL of DM fermentation medium, and lactose was added to a final concentration of 3 g / L. The culture was then continued at 30°C, 200 rpm, for 72 hours. The supernatant (1 mL) of fermentation broth was collected and the 6′-sialyllactose content was determined by HPLC. The results are shown in Table 8.

[0238] Table 7

[0239]

[0240]

[0241] Table 8

[0242]

[0243] Example 7: Efficient production of 6'-sialyllactose in an engineered bacterial fermenter

[0244] In order to further verify the effectiveness of the 6'-sialyllactose synthesis method and improve the yield of 6'-sialyllactose, the engineered bacteria EM33MS seed solution was inoculated into a 2L DM fermentation medium at a 7.5% (v / v) inoculation rate, and the initial temperature was maintained at 37°C. When the OD 600 When the temperature reaches 30-33°C, cool it down to 25°C and add lactose with a final concentration of 2g / L. Then, feed the fermentation liquid to ensure that the glycerol is maintained at a final concentration of 2-8g / L and the lactose is maintained at a final concentration of 2-5g / L. The pH is kept at 6.8±0.1 throughout the process, and the foam is controlled by adding a defoamer. The dissolved oxygen is controlled at 20%-30% by adjusting the stirring speed and ventilation volume. After the whole process of cultivation reaches 60h, the bacterial OD 600 The extracellular production of 6'-sialyllactose reached the highest level, reaching 14.08 g / L (e.g. Figure 4 ).

[0245] Example 8: Construction of a plasmid-free strain with high 6'-sialyllactose production based on Hmp0133 enzyme in K12-MG1655 host strain

[0246] In this example, MAK was used as the starting strain, and different copies of the exogenous tandem gene neuBCA, a novel exogenous α2,6-sialyltransferase, and an enhanced precursor gene were introduced to significantly increase the production of 6'-sialyllactose.

[0247] The specific strain transformation method is described in Example 3. One or more copies of the constitutive promoter P are integrated into the manXYZ, arsB, and curA sites of the MAK host bacterial genome. J23119 Under the control of neuBCA, one or more copies of the constitutive promoter P are integrated at the poxB, yghWX, and ldhA sites. J23119 Under the control of α2,6-sialyltransferase Hmp0133, engineered bacteria HM11, HM12, HM21, HM22, HM23, and HM33 were obtained.

[0248] A copy of the constitutive promoter P was integrated into the adhE site of the engineered bacterium HM23 genome. J23119 The engineered bacteria HM23M were obtained by controlling glmM.

[0249] A copy of the constitutive promoter P was integrated into the xylB site of the engineered bacterium HM23M genome. J23119 The engineered bacteria HM23MS were obtained by expressing glmS under its control.

[0250] A copy of the constitutive promoter P was integrated into the fuci site of the engineered bacterium HM23MS genome. J23119 The engineered bacteria HM23MSU were obtained under the control of glmU.

[0251] in,

[0252] The amino acid sequence of glmU is shown in SEQ ID NO. 33, and the nucleotide sequence is shown in SEQ ID NO. 34;

[0253] The primers involved in the above method are shown in Table 9, the engineering bacteria information is shown in Table 10, and the rest is the same as Example 3.

[0254] Production of 6'-sialyllactose by shake flask fermentation:

[0255] Each of the nine engineered strains constructed above was inoculated into LB liquid medium and cultured overnight at 37°C, 200 rpm, for 12 hours to obtain seed liquid. A 2% portion of the seed liquid was inoculated into 25 mL of DM fermentation medium. Lactose was added to a final concentration of 3 g / L and cultured at 30°C, 200 rpm, for 72 hours. The supernatant (1 mL) of fermentation broth was collected and the 6′-sialyllactose content was determined by HPLC. The results are shown in Table 10.

[0256] Table 9

[0257]

[0258] Table 10

[0259]

[0260]

[0261] Example 9: Efficient production of 6'-sialyllactose in an engineered bacterial fermenter

[0262] In order to further verify the effectiveness of the 6'-sialyllactose synthesis method and improve the yield of 6'-sialyllactose, the engineered bacteria HM23MS seed solution was inoculated into a 2L DM fermentation medium at a 7.5% (v / v) inoculation rate, and the initial temperature was maintained at 37°C. When the OD 600 When the temperature reaches 30-33°C, cool it down to 25°C and add lactose with a final concentration of 2g / L. Then, feed the fermentation feed solution to ensure that the glycerol concentration is maintained at 2-8g / L and the lactose concentration is maintained at 2-5g / L. The pH is kept at 6.8±0.1 throughout the process, and the foam is controlled by adding a defoamer. The dissolved oxygen is controlled at 20%-30% by adjusting the stirring speed and ventilation volume. After the whole process of cultivation reaches 54h, the bacterial OD 600 The extracellular production of 6'-sialyllactose reached the highest level, reaching 17.22 g / L (e.g. Figure 5 ).

[0263] The foregoing is merely 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 modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. The use of a protein in constructing an engineered bacterium that produces 6'-sialyllactose, preparing 6'-sialyllactose, or regulating the production of 6'-sialyllactose by a microorganism, characterized in that: The protein includes at least any one or more of the following A1)-A9): A1) Glycosyltransferase from Kingella denitrificans; A2) glycosyltransferase from Vespertiliibacter pulmonis; A3) Glycosyltransferase from Otariodibacter oris; A4) Glycosyltransferase from Paramixta manurensis; A5) Glycosyltransferase from Pseudoalteromonas rubra; A6) glycosyltransferase from Nicoletella semolina; A7) Glycosyltransferase from Muribacter muris; A8) glycosyltransferase from Simonsiella muelleri; A9) Glycosyltransferase from Pasteurellaceae bacterium.

2. The use according to claim 1, characterized in that The protein includes at least any one or more of the following B1)-B11): B1) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 8; B2) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 10; B3) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 12; B4) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 14; B5) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 16; B6) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 18; B7) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 20; B8) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 22; B9) a glycosyltransferase comprising the amino acid sequence of SEQ ID NO. 24; B10) contains a protein having 99% or greater identity with the proteins defined in B1) to B9) and associated with 6'-sialyllactose production; B11) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of B1) to B10).

3. Biomaterial, characterized in that The biological material includes at least one of the following C1) to C3): C1) a nucleic acid molecule encoding the protein according to claim 1 or 2; C2) an expression cassette containing the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1) and / or a recombinant vector containing the expression cassette described in C2).

4. The biomaterial according to claim 3, characterized in that: The nucleic acid molecule described in C1) includes any one or more of D1) to D10): D1) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 9; D2) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 11; D3) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 13; D4) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 15; D5) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 17; D6) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 19; D7) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 21; D8) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 23; D9) a nucleic acid molecule whose nucleotide sequence comprises SEQ ID NO. 25; D10) A nucleic acid molecule having 80% or greater identity with the nucleic acid molecule according to any one of D1) to D9).

5. A recombinant microorganism, characterized in that The recombinant microorganism includes any one or more of the following E1)-E4): E1) a recombinant microorganism containing the protein according to claim 1 or claim 2; E2) a recombinant microorganism containing the nucleic acid molecule according to C1) of claim 3; E3) a recombinant microorganism containing the expression cassette according to C2) of claim 3; E4) A recombinant microorganism containing the recombinant vector described in C3) of claim 3.

6. The recombinant microorganism according to claim 5, characterized in that The recombinant microorganism further contains a gene for expressing a precursor enzyme; Preferably, the precursor enzyme is neuBCA derived from Campylobacter jejuni.

7. The recombinant microorganism according to claim 6, characterized in that The expression ratio of the precursor enzyme to the protein according to claim 1 in the recombinant microorganism is (1-2):

1.

8. The recombinant microorganism according to any one of claims 5 to 7, characterized in that The recombinant microorganism is any one of the following F1)-F2): F1) Escherichia bacteria; F2) Escherichia coli.

9. A whole-cell catalyst comprising the recombinant microorganism according to any one of claims 5 to 8.

10. A method for preparing 6'-sialyllactose, characterized in that: The method comprises: preparing 6'-sialyllactose by fermentation using the biological material according to claim 3 or 4, the recombinant microorganism according to any one of claims 5 to 8, or the whole-cell catalyst according to claim 9.

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