Proteome, multienzyme complex and application thereof, and preparation method of sialic acid lactose

Abstract

The invention relates to the field of bioengineering, in particular to a proteome, a multi-enzyme complex, application of the proteome and the multi-enzyme complex and a preparation method of sialic acid lactose. The invention provides an application of a proteome in preparation of sialic acid lactose. The proteome comprises a first protein and a second protein; the first protein comprises any two of SpyTag, SnoopTag, SdyTag or RIAD (Ribonucleic Acid Discriminant); the second protein comprises any two of a SpyCatcher, a SnoopCatcher, a SdyCatcher or an RIDD (Rich Independent Disk Discriminant); the first protein and the second protein are covalently bound to form a protein interaction pair. According to the method disclosed by the invention, ordered assembly of multiple enzymes in a two-dimensional space is realized through two protein interaction pairs with orthogonality, and efficient preparation of sialic acid lactose is achieved. According to the invention, CMP kinase and polyphosphate kinase are subjected to fusion expression, the enzyme dosage required by the process is reduced, and meanwhile, sufficient CTP supply is ensured.

Description

Technical Field

[0001] The invention relates to the field of bioengineering, and in particular to a protein group, a multi-enzyme complex and application thereof, and a preparation method of sialyllactose. Background Art

[0002] Human milk oligosaccharides (HMO) is a general term for all oligosaccharides in human milk. It is the second largest carbohydrate component and the third largest nutrient in breast milk after lactose, accounting for 10% of the dry matter of breast milk. HMOs have significant beneficial functions for the healthy growth and development of infants and young children. They are not digested and absorbed in the human body, but directly reach the intestines, can selectively promote the growth of beneficial bacteria in the intestines, indirectly inhibit the growth of harmful bacteria, and maintain the balance of intestinal microecology. At the same time, HMOs can also regulate the immune system, stimulate the body to produce immune active substances, and thus enhance the body's immune capacity. At present, HMOs are mainly divided into three categories: fucosylated HMOs, non-fucosylated HMOs and sialylated HMOs. Among them, sialylo-oligosaccharides are important components of human milk oligosaccharides, which are present in large quantities in colostrum (1-3.3 g / L). According to the position where sialic acid is combined with lactose, it can be divided into 3'-sialyllactose (3'-sialyllactose, 3'-SL) or 6'-sialyllactose (6'-sialyllactose, 6'-SL). Many studies have shown that sialyllactose has important functions such as resisting pathogen infection, promoting intestinal maturation, improving immune function, and enhancing brain cognitive development. Due to the unique structure and negative charge of sialic acid, its physiological function cannot be completely replaced by other oligosaccharides, and sialyllactose has gradually shown superior market potential.

[0003] At present, the preparation methods of sialyllactose include chemical synthesis, biological fermentation and enzyme catalysis. The chemical method synthesizes sialyllactose through automatic solid phase, and its reaction conditions are relatively harsh. It is necessary to repeatedly carry out selective protection and deprotection of functional groups. The preparation process is cumbersome, the environmental cost is high, and it is difficult to ensure the stereoselectivity of glycosidic bonds. The fermentation method for preparing sialyllactose has a long fermentation cycle, and it is also easy to cause the product to accumulate in the cell, resulting in feedback inhibition, low yield, high production cost, and also cannot meet the needs of industrialization. The preparation of sialyllactose by enzyme method has long been reported in the literature. Exo-α-sialidase and sialyltransferase can catalyze the one-step synthesis of sugar chains. The former transfers the donor sialic acid to lactose to form sialyllactose. This catalytic reaction does not require an additional energy donor, but exo-α-sialidase can only recognize the donor's α-2,3-bound sialic acid, and the regional selectivity is not strict. It usually synthesizes α-2,3-sialic acid oligosaccharide isomers, the products are difficult to separate, and the product conversion rate is low. Sialyltransferase can also transfer sialic acid monomers to lactose, but sialic acid must be in an active form, such as cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-SA). However, activated sialic acid is often expensive, which seriously restricts the large-scale production of sialyllactose. To solve the above problems, SA can be selected as a substrate, and activated intermediates can be prepared under the action of CMP-sialic acid synthetase. In combination with the CTP regeneration system, 3'-SL and 6'-SL can be synthesized under the action of the corresponding sialyltransferase. For example, Gilbert et al. used lactose, N-acetylneuraminic acid, phosphoenolpyruvate, ATP and CMP as raw materials, and used the fusion enzyme of CMP-SA synthase and α-2,3-sialyltransferase to obtain 3'-SL. Li Zhongkui et al. used the CTP cycle system to catalyze the corresponding substrates using CMP kinase, polyphosphate kinase, CMP-SA synthetase and sialyltransferase to obtain sialyllactose in one pot.

[0004] However, the above-mentioned multi-enzyme method for catalyzing the synthesis of sialyllactose is prone to long catalytic time and low yield. This is because in most cases, the enzymes in the system exist in the form of free enzymes, there is steric hindrance or excessive dispersion between the enzyme molecules, and the intermediates are evenly diffused in the solution, which makes it difficult to transfer the intermediates, thereby significantly reducing the synergy of the multi-enzyme catalytic reaction. In recent years, inspired by natural multi-enzyme complexes, the construction of artificial multi-enzyme complexes has begun to attract much attention. Carrying out cascade catalytic reactions in the form of artificial multi-enzyme complexes is conducive to the formation of substrate channels, which can prevent the diffusion of intermediates, shorten the transfer time from one active site to another, protect intermediates from the influence of competitive enzyme reactions, avoid the decomposition of unstable intermediates, reduce unfavorable reaction balance, and improve the overall catalytic efficiency of cascade enzymes. The skeleton-free self-assembly technology based on protein interaction pairs is currently a popular multi-enzyme assembly method. It effectively overcomes the deficiencies of traditional gene fusion technology and protein skeleton system in the construction of multi-enzyme complexes to a certain extent, and completes the orderly assembly of multiple enzyme molecules. At present, there are few reports on the application of skeleton-free self-assembly technology in the enzymatic synthesis of sialyllactose. Summary of the invention

[0005] In view of this, the present invention provides a protein group, a multi-enzyme complex and its application and a method for preparing sialyllactose. This patent adopts a method for constructing a multi-enzyme complex to achieve efficient preparation of sialyllactose. This patent fuses CMP kinase and polyphosphate kinase for expression, which not only ensures sufficient supply of CTP, but also reduces enzyme dosage, saves production costs, and innovatively applies multiple protein interactions to orderly assemble CMP-SA synthase, CMP kinase-polyphosphate kinase fusion enzyme and sialyltransferase, constructs a substrate channel, promotes the conversion of intermediates, and obtains sialyllactose from sialic acid in a one-pot method, achieving high substrate conversion rate (>87%) and high product concentration (>110g / L) in a short time.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an application of a protein group in preparing sialyllactose; the protein group comprises a first protein and a second protein;

[0008] The first protein includes any two of SpyTag, SnoopTag, SdyTag or RIAD;

[0009] The second protein includes any two of SpyCatcher, SnoopCatcher, SdyCatcher or RIDD;

[0010] The first protein and the second protein are covalently bound to form a protein interaction pair.

[0011] In some embodiments of the present invention, in the above applications, the protein interaction pair includes: SpyTag / SpyCatcher, SnoopTag / SnoopCatcher, SdyTag / SdyCatcher or RIAD / RIDD.

[0012] The present invention also provides a recombinant expression vector composition, including: a CSS recombinant expression vector, a CMK-PPK recombinant expression vector and a ST recombinant expression vector;

[0013] The CSS recombinant expression vector is obtained by coupling CSS with the first protein-1 and the first protein-2 respectively;

[0014] The CMK-PPK recombinant expression vector is obtained by fusing CMK and PPK and coupling with the second protein-1;

[0015] The ST recombinant expression vector is obtained by coupling ST with a second protein-2;

[0016] The first protein-1 and the first protein-2 are independently selected from the first protein and / or the second protein in the above application;

[0017] The first protein-1 and the first protein-2 are not identical;

[0018] The second protein-1 and the second protein-2 are independently selected from the first protein and / or the second protein in the above application;

[0019] The second protein-1 and the second protein-2 are not identical;

[0020] The second protein-1 is covalently bound to the first protein-1 to form the protein interaction pair in the above application;

[0021] The second protein-2 is covalently bound to the first protein-2 to form the protein interaction pair in the above application.

[0022] In some embodiments of the present invention, in the above-mentioned recombinant expression vector composition, the ST comprises: 3-ST or 6-ST.

[0023] In some embodiments of the present invention, in the above-mentioned recombinant expression vector composition, the CSS recombinant expression vector comprises: one or more of SpyTag-CSS-SnoopTag plasmid, SpyTag-CSS-RIAD plasmid, RI AD-CSS-SdyTag plasmid, SpyTag-CSS-SdyTag plasmid and / or SnoopTag-CSS-SpyTag plasmid;

[0024] The CMK-PPK recombinant expression vector comprises: one or more of a CMK-PPK-SpyCatcher plasmid, a CMK-PPK-RIDD plasmid, a CMK-PPK-SdyCatcher plasmid and / or a SpyCatcher-CMK-PPK plasmid;

[0025] The ST recombinant expression vector comprises: one or more of 3-ST-SnoopCatcher plasmid, 3-ST-SpyCatcher plasmid, SdyCatcher-3-ST plasmid, SdyCatcher-6-ST plasmid, 6-ST-SpyCatcher plasmid and / or 6-ST-SnoopCatcher plasmid.

[0026] In some embodiments of the present invention, the CSS in the above-mentioned recombinant expression vector composition has:

[0027] (1), the nucleotide sequence shown in SEQ ID NO: 1; or

[0028] (2) a nucleotide sequence obtained by substituting, deleting or adding one or more bases to the nucleotide sequence shown in (1), and having the same or similar function as the nucleotide sequence shown in (1); or

[0029] (3) a nucleotide sequence having at least 80% identity with the nucleotide sequence shown in (1) or (2); and

[0030] The CMK has:

[0031] (4) the nucleotide sequence shown in SEQ ID NO: 2; or

[0032] (5) a nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as the nucleotide sequence shown in (4); or

[0033] (6) a nucleotide sequence having at least 80% identity with the nucleotide sequence shown in (4) or (5); and

[0034] The PPK has:

[0035] (7), the nucleotide sequence shown in SEQ ID NO: 3; or

[0036] (8) a nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (7), and having the same or similar function as the nucleotide sequence shown in (7); or

[0037] (9) a nucleotide sequence having at least 80% identity with the nucleotide sequence shown in (7) or (8); and

[0038] The ST has:

[0039] (10), the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5; or

[0040] (11) a nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (10), and having the same or similar function as the nucleotide sequence shown in (10); or

[0041] (12) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (10) or (11).

[0042] In some embodiments of the present invention, the nucleotide sequence of the CSS in the above recombinant expression vector composition is as shown in SEQ ID NO: 1: ATGAAGATCATTATGACTCGTATTGCAA TTATTCCTGCACGGGCTGGCTCGAAGGGTATTAAAGATAAGAACTTGCAACTCGTTGGCGGCGTTTCACTTGTAGGTCGTGCCATTTTGGCAGCACAGGAATCAGGCATGTTCGACCAAATCGTTGTGACTTCCGACGGCGAGAATATCTTGAAGGAGGCTACGAAGTATGGGGCTAAACCGGTAGCGCGTCCAGAGTCGCTGGCGCAATCAGATACCCGGACGATCGATGCGATTCTGCATTGTTTGGAAACACTTAACATTTCTCAAGGGACGGCTGCCTTGCTGCAGCCAACCAGCCCACTTCGGAACGCTCTGGACATTCGTAACGCAATGGAGATCTTCCTCGGTGGGAAATACAAGAGCGTCGTGTCTGCATGCGAGTGTGAACATCACCCTTATAAGTCGTTCACTTTGGAGGGGACTGAGGTACAGCCTATCCATGAACTCACGGACTTTGAATCGCCGCGTCAAAAGTTGCCTAAATCTTATCGTGCGAATGGGGCTATCTACATCAATGACATCCAGTCTCTCTTCGAGGAAAAACGCTTCTTCATCGCCCCAATGCGTTTCTATCTCATGCCGACATATCGCTCGATTGACATCGATTCCACATTAGATCTCCAACTGGCAGAGTCCCTGATTAGTAAGGAGTTCTAA。

[0043] In some embodiments of the present invention, the nucleotide sequence of CMK in the above recombinant expression vector composition is as shown in SEQ ID NO:2: ATGAACATCGTTCCGGTTATTACCCTGG ATGGCCCGGGTGGCGTGGGTAAAGGTACCCTGGGTCGTCTGCTGGCCCATGATCTGGGCTGGCATCTGCTGGATAGCGGTGCAATCTATCGTGCCCTGGCACTGGCCGCACAGCGTGCAGGTCTGGATGCAGATAATGAAACCGCACTGAGTGGTCTGGCACGCACCCTGCCGCTGGGCTTCCGTGGTGCAGCCGATGAAACACATGTTCTGCTGGGCGGTGAAGTTGTGGATGCAGAAATTCGTACCCCGGAAGTGAGCGCCCTGACCAGCCGTATTGCAGCCATTCCGGCCGTTCGCGCAGCCCTGCTGCAGAGACAGCGCGACTTCCGCCGCGCACCGGGTCTTGTGGCCGATGGTCGCGATATGGGTACCGTTGTGTTCCCGGATGCACCGCTGAAAGTGTTCCTGACCGCCAGTGCAGAAGAACGTGGTAAACGCCGTCTGAATCAGCTGATGGAACAGGGTATTAGCGCAAGTCTGGCCACCGTGGTTGCCGAAATTGCAGAACGTGATGCACGTGATCAGCAGCGTAGTGTTGCACCGCTGCGTCCGGCCCCGGATGCTAAAATTCTGGATACCAGTGAACAGAGCGTTAGTGATACCTATCGTGTGCTGCAGAGTTGGGTGCGCAGTGCCTTCCAGGAAAATTAA。

[0044] In some embodiments of the present invention, the nucleotide sequence of the PPK in the above recombinant expression vector composition is as shown in SEQ ID NO:3: ATGGCCCGTAATGGTAATGGTCGTAAA GATGCCGCAGGCAAAGCAAAAAGCCTGCCGGATCTGCCGTTCGTGGGTGCAATTACCCGCTATCTGGAAAAAGATGCCCCGGAAGCCCTGCGCAAAGCAGTTGCCGCAGCAGATGGTAAACGCATTCTGGATCCGAGTTATCCGCATGCAGAAGAAATGAAAGGTAAAGATTATGATGACCGCATGGCCGGTCTGCAGCTGCAGCTGGTGCGTCTGATGCGTGATGTTATTCATACCGGCAAACGTATGGTGGTGCTGTTCGAAGGTCGTGATGCCGCCGGTAAAGGTGGCACCATTGAACGCGTGCGTGAAAATCTGAATCCGCGCAGTGCATATATTGTGGCACTGCCGAAACCGAGTGAACGTGAAGCCGAAGAATGGTACTTCCAGCGTTATGTTGATTGGCTGCCGGCAAAAGGTGAAATTGCACTGTTCGATCGCAGCTGGTATAATCGTGGTGTTGTGGAAAAAGTGTTCGGCTTCTGCACCGATGCCCAGCGCGAAACCTTCTTCCGCCAGCTGCCGCTGTTCGAAAATATGCTGGTTGATGATGGTGTGATTCTGGTGAAACTGTGGCTGGAAGTTGGCCGCGCCGAACAGCTGAAACGCTTCCTGGATCGTGAACAGGATCCGCTGAAACAGTGGAAACTGAGTCCGATTGATGTGGAAGGTCTGGCAAAATGGGAAGATTATAGCGCAGCCATTCAGGATACCCTGCTGCGTAGCCATAGCGGCATTGCCCCGTGGACCGTGATTCGTGCAGATGATAAACGTCGCGCACGTCTGGCCGCCATTCAGACCATTCTGCGCGCCGTTGACTTCGCCGGCCGCGATGATGAAGTTATTGGTCAGCCGGATCCGCAGATTACCGGCGGTCCGGAAATTCTGCCGGGCTAA。

[0045]

[0046]

[0047] In some embodiments of the present invention, the nucleotide sequence of the SpyTag-CSS-SnoopTag plasmid in the above recombinant expression vector composition is as shown in SEQ ID NO:6: ATGGCGCATATTG TGATGGTGGATGCGTATAAACCGACCAAAGGAGGCGGTGGGAGTGGAGGCGGTGGAAGTGGCGGAGGTGGCTCTATGAAGATCATTATGACTCGTATTGCAATTATTCCTGCACGGGCTGGCTCGAAGGGTATTAAAGATAAGAACTTGCAACTCGTTGGCGGCGTTTCACTTGTAGGTCGTGCCATTTTGGCAGCACAGGAATCAGGCATGTTCGACCAAATCGTTGTGACTTCCGACGGCGAGAATATCTTGAAGGAGGCTACGAAGTATGGGGCTAAACCGGTAGCGCGTCCAGAGTCGCTGGCGCAATCAGATACCCGGACGATCGATGCGATTCTGCATTGTTTGGAAACACTTAACATTTCTCAAGGGACGGCTGCCTTGCTGCAGCCAACCAGCCCACTTCGGAACGCTCTGGACATTCGTAACGCAATGGAGATCTTCCTCGGTGGGAAATACAAGAGCGTCGTGTCTGCATGCGAGTGTGAACATCACCCTTATAAGTCGTTCACTTTGGAGGGGACTGAGGTACAGCCTATCCATGAACTCACGGACTTTGAATCGCCGCGTCAAAAGTTGCCTAAATCTTATCGTGCGAATGGGGCTATCTACATCAATGACATCCAGTCTCTCTTCGAGGAAAAACGCTTCTTCATCGCCCCAATGCGTTTCTATCTCATGCCGACATATCGCTCGATTGACATCGATTCCACATTAGATCTCCAACTGGCAGAGTCCCTGATTAGTAAGGAGTTCGGGGGGAGTGGCGGCTCTGGGGGCTCTGAGGGAGGCTCAGGTGGGTCGATGGGCAAACTGGGCGATATTGAATTTATTAAAGTGAACAAATAA。

[0048] In some embodiments of the present invention, the nucleotide sequence of the SpyTag-CSS-RIAD plasmid in the above recombinant expression vector composition is as shown in SEQ ID NO:7: ATGGCGCATATTGTGATGGTGGATGCGTATAAACCGACCAAAGGAGGCGGTGGGAGTGGAGGCGGTGGAAGTGGCGGAGGTGGCTCTATGAAGATCATTATGACTCGTATTGCAATTATTCCTGCACGGGCTGGCTCGAAGGGTATTAAAGATAAGAACTTGCAACTCGTTGGCGGCGTTTCACTTGTAGGTCGTGCCATTTTGGCAGCACAGGAATCAGGCATGTTCGACCAAATCGTTGTGACTTCCGACGGCGAGAATATCTTGAAGGAGGCTACGAAGTATGGGGCTAAACCGGTAGCGCGTCCAGAGTCGCTGGCGCAATCAGATACCCGGACGATCGATGCGATTCTGCATTGTTTGGAAACACTTAACATTTCTCAAGGGACGGCTGCCTTGCTGCAGCCAACCAGCCCACTTCGGAACGCTCTGGACATTCGTAACGCAATGGAGATCTTCCTCGGTGGGAAATACAAGAGCGTCGTGTCTGCATGCGAGTGTGAACATCACCCTTATAAGTCGTTCACTTTGGAGGGGACTGAGGTACAGCCTATCCATGAACTCACGGACTTTGAATCGCCGCGTCAAAAGTTGCCTAAATCTTATCGTGCGAATGGGGCTATCTACATCAATGACATCCAGTCTCTCTTCGAGGAAAAACGCTTCTTCATCGCCCCAATGCGTTTCTATCTCATGCCGACATATCGCTCGATTGACATCGATTCCACATTAGATCTCCAACTGGCAGAGTCCCTGATTAGTAAGGAGTTCGGGGGGAGTGGCGGCTCTGGGGGCTCTGAGGGAGGCTCAGGTGGGTCGATGCTGGAGCAGTACGCAAACCAGTTAGCGGACCAGATTATTAAAGAGGCTACGGAATAA。

[0049] In some embodiments of the present invention, the nucleotide sequence of the RIAD-CS S-SdyTag plasmid in the above recombinant expression vector composition is as shown in SEQ ID NO:8: ATGCTGGAGCAGTAC GCAAACCAGTTAGCGGACCAGATTATTAAAGAGGCTACGGAAGGAGGCGGTGGGAGTGGAGGCGGTGGAAGTGGCGGAGGTGGCTCTATGAAGATCATTATGACTCGTATTGCAATTATTCCTGCACGGGCTGGCTCGAAGGGTATTAAAGATAAGAACTTGCAACTCGTTGGCGGCGTTTCACTTGTAGGTCGTGCCATTTTGGCAGCACAGGAATCAGGCATGTTCGACCAAATCGTTGTGACTTCCGACGGCGAGAATATCTTGAAGGAGGCTACGAAGTATGGGGCTAAACCGGTAGCGCGTCCAGAGTCGCTGGCGCAATCAGATACCCGGACGATCGATGCGATTCTGCATTGTTTGGAAACACTTAACATTTCTCAAGGGACGGCTGCCTTGCTGCAGCCAACCAGCCCACTTCGGAACGCTCTGGACATTCGTAACGCAATGGAGATCTTCCTCGGTGGGAAATACAAGAGCGTCGTGTCTGCATGCGAGTGTGAACATCACCCTTATAAGTCGTTCACTTTGGAGGGGACTGAGGTACAGCCTATCCATGAACTCACGGACTTTGAATCGCCGCGTCAAAAGTTGCCTAAATCTTATCGTGCGAATGGGGCTATCTACATCAATGACATCCAGTCTCTCTTCGAGGAAAAACGCTTCTTCATCGCCCCAATGCGTTTCTATCTCATGCCGACATATCGCTCGATTGACATCGATTCCACATTAGATCTCCAACTGGCAGAGTCCCTGATTAGTAAGGAGTTCGGGGGGAGTGGCGGCTCTGGGGGCTCTGAGGGAGGCTCAGGTGGGTCGATGGATCCGATTGTGATGATTGATAACGATAAACCGATTACCTAA。

[0050] In some embodiments of the present invention, the nucleotide sequence of the SpyTag-CSS-SdyTag plasmid in the above recombinant expression vector composition is as shown in SEQ ID NO:9: ATGGCGCATATTGTG ATGGTGGATGCGTATAAACCGACCAAAGGAGGCGGTGGGAGTGGAGGCGGTGGAAGTGGCGGAGGTGGCTCTATGAAGATCATTATGACTCGTATTGCAATTATTCCTGCACGGGCTGGCTCGAAGGGTATTAAAGATAAGAACTTGCAACTCGTTGGCGGCGTTTCACTTGTAGGTCGTGCCATTTTGGCAGCACAGGAATCAGGCATGTTCGACCAAATCGTTGTGACTTCCGACGGCGAGAATATCTTGAAGGAGGCTACGAAGTATGGGGCTAAACCGGTAGCGCGTCCAGAGTCGCTGGCGCAATCAGATACCCGGACGATCGATGCGATTCTGCATTGTTTGGAAACACTTAACATTTCTCAAGGGACGGCTGCCTTGCTGCAGCCAACCAGCCCACTTCGGAACGCTCTGGACATTCGTAACGCAATGGAGATCTTCCTCGGTGGGAAATACAAGAGCGTCGTGTCTGCATGCGAGTGTGAACATCACCCTTATAAGTCGTTCACTTTGGAGGGGACTGAGGTACAGCCTATCCATGAACTCACGGACTTTGAATCGCCGCGTCAAAAGTTGCCTAAATCTTATCGTGCGAATGGGGCTATCTACATCAATGACATCCAGTCTCTCTTCGAGGAAAAACGCTTCTTCATCGCCCCAATGCGTTTCTATCTCATGCCGACATATCGCTCGATTGACATCGATTCCACATTAGATCTCCAACTGGCAGAGTCCCTGATTAGTAAGGAGTTCGGGGGGAGTGGCGGCTCTGGGGGCTCTGAGGGAGGCTCAGGTGGGTCGATGGATCCGATTGTGATGATTGATAACGATAAACCGATTACCTAA。

[0051]

[0052]

[0053]

[0054] In some embodiments of the present invention, the nucleotide sequence of the CMK-PP K-SdyCatcher plasmid in the above recombinant expression vector composition is as shown in SEQ ID NO: 13: ATGAACATCGTT CCGGTTATTACCCTGGATGGCCCGGGTGGCGTGGGTAAAGGTACCCTGGGTCGTCTGCTGGCCCATGATCTGGGCTGGCATCTGCTGGATAGCGGTGCAATCTATCGTGCCCTGGCACTGGCCGCACAGCGTGCAGGTCTGGATGCAGATAATGAAACCGCACTGAGTGGTCTGGCACGCACCCTGCCGCTGGGCTTCCGTGGTGCAGCCGATGAAACACATGTTCTGCTGGGCGGTGAAGTTGTGGATGCAGAAATTCGTACCCCGGAAGTGAGCGCCCTGACCAGCCGT

[0055] ATTGCAGCCATTCCGGCCGTTCGCGCAGCCCTGCTGCAGAGACAGCGC

[0056] GACTTCCGCCGCGCACCGGGTCTTGTGGCCGATGGTCGCGATATGGGTA

[0057] CCGTTGTGTTCCCGGATGCACCGCTGAAAGTGTTCCTGACCGCCAGTGC

[0058] AGAAGAACGTGGTAAACGCCGTCTGAATCAGCTGATGGAACAGGGTAT

[0059] TAGCGCAAGTCTGGCCACCGTGGTTGCCGAAATTGCAGAACGTGATGC

[0060] ACGTGATCAGCAGCGTAGTGTTGCACCGCTGCGTCCGGCCCCGGATGCT

[0061] AAAATTCTGGATACCAGTGAACAGAGCGTTAGTGATACCTATCGTGTG

[0062] CTGCAGAGTTGGGTGCGCAGTGCCTTCCAGGAAAATGGGGGGAGTGGC

[0063] GGCTCTGGGGGCTCTGAGGGAGGCTCAGGTGGGTCGATGGCCCGTAAT

[0064] GGTAATGGTCGTAAAGATGCCGCAGGCAAAGCAAAAAGCCTGCCGGAT

[0065] CTGCCGTTCGTGGGTGCAATTACCCGCTATCTGGAAAAAGATGCCCCGG

[0066] AAGCCCTGCGCAAAGCAGTTGCCGCAGCAGATGGTAAACGCATTCTGG

[0067] ATCCGAGTTAATCCGCATGCAGAAGAATGAAAGGTAAAGATTATGATG

[0068] ACCGCATGGCCGGTCTGCAGCTGCAGCTGGTGCGTCTGATGCGTGATGT

[0069] TATTCATACCGGCAAACGTATGGTGGTGCTGTTCGAAGGTCGTGATGCC

[0070] GCCGGTAAAGGTGGCACCATTGAACGCGTGCGTGAAAATCTGAATCCG

[0071] CGCAGTGCATATATTGTGGCACTGCCGAAACCGAGTGAACGTGAAGCC

[0072] GAAGAATGGTACTTCCAGCGTTATGTTGATTGGCTGCCGGCAAAAGGT

[0073] GAAATTGCACTGTTCGATCGCAGCTGGTAATCGTGGTGTTGTGGAAA

[0074] AAGTGTTCGGCTTCTGCACCGATGCCCAGCGCGAACCTTCTTCCGCCA

[0075] GCTGCCGCTGTTCGAAAATATGCTGGTTGATGATGGTGTGATTCTGGTG

[0076] AAACTGTGGCTGGAAGTTGGCCGCGCCGAACAGCTGAAACGCTTCCTG

[0077] GATCGTGAACAGGATCCGCTGAAACAGTGGAAACTGAGTCCGATTGAT

[0078] GTGGAAGGTCTGGCAAAATGGGAAGATTATAGCGCAGCCATTCAGGAT

[0079] ACCCTGCTGCGTAGCCATAGCGGCATTGCCCCGTGGACCGTGATTCGTG

[0080] CAGATGATAAACGTCGCGCACGTCTGGCCGCCATTCAGACCATTCTGCG

[0081] CGCCGTTGACTTCGCCGGCCGCGATGATGAAGTTATTGGTCAGCCGGAT

[0082] CCGCAGATTACCGGCGGTCCGGAAATTCTGCCGGGCGGGGGTGGTGGC

[0083] TCAGAGGCGGCGAAAGAAGCCGCCAAAGGTGGAGGAGGATCTATGGG

[0084] CAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGG

[0085] CAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATC

[0086] CTCCGGTCTGAGCGGTGAGACGGGTCAGTCCGGCAATACCACTATCGA

[0087] AGAGGACAGCACCACTCATGTTAAATTCTCCAAACGTGACGCGAACGG

[0088] TAAAGAACTGGCTGGCGCCATGATTGAACTGCGTAACCTGAGCGGCCA

[0089] GACTATTCAGTCCTGGATTTCTGACGGCACCGTCAAAGTTTTCTACCTG

[0090] ATGCCAGGCACCTATCAGTTCGTTGAAACCGCGGCGCCGGAAGGCTAC

[0091] GAGCTGGCCGCCCCGATTACCTTCACTATCGACGAGAAAGGCCAGATCTGGGTGGACAGCTAA。

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] In some embodiments of the present invention, the nucleotide sequence of the 6-ST-Spy Catcher plasmid in the above-mentioned recombinant expression vector composition is as shown in SEQ ID NO: 19: ATGCGCATCCTCTTTGT GGTCGCATTTTTCGCTTCATTGGCTGCCGTCTGTCAAGCGCAATCGTCGCTCCAAGCGGAGGTTCTTCAAAAGCAGAATCCAGTACTTGTTGGTGTAAAGAACAATGTTGCCCTCTGCCTGAAGGTTAGCGTTCCTGAGAACCAACAGATTGCAATTAGTGAACTCCGTTTCAGTACAAAGGGTACGTCACGCCTTTCCGACTTGAAGGCTTTCCGGGTTTTTTCGACAGGGACTCGCAGCAGT

[0098] TTCGTAGACTCCGCCATGGTCTCGCAAACTGCTAAGGTCGGTAAAGAA

[0099] CTTGCCGTAAAGGAGGCGATTATGTTACAGAAAGGGGAGAACTATTTG

[0100] TGGGTGACACTCGAATTGAAGGATAAAGTGAACTTGCTTAATCGTTTCG

[0101] ATGTTGCTTGCTCGTCGATGACTCTCTCTAATGGCGCGGTCTTGACACC

[0102] AACAGATCGGACACCAAATGTCGTATTACGGGCAGGCTACGCCGTTCG

[0103] TCAACGGATGCAAGACGGGGTCAATACCTACCGTATTCCAGGTTTAGTC

[0104] ACCACTAAAAAAGGGACATTAATTTCGGTTTTCGATATTCGTCGGAAAT

[0105] ACTCCGGTGACCTTCAAGGTGACATCGATATCGGGATCTGTCGCTCAAT

[0106] GGACGGGGGCAAAAGTTGGCTCCCAATGCAAGTCGCGATTGATATGGG

[0107] GGAATGGGGGGGCCTCTCCCATGACGAAAATGGTATTACTGATCCAAG

[0108] CATCCTGGTGGATGAGACCACGGGGACTGTTTGGATTGCTGCTTTATGG

[0109] CTTAACGGCTCGAAAGGGAAGCCGGCATGGTGGGCTTCGAAGCCTGGG

[0110] ATGACTCCGGATGTAACCGGCCAGTTCGTATTAGCCAAATCGGATGAC

[0111] GATGGCTTGACATGGTCGGAGCCTATCAATGTAACTGAACAAATGAAA

[0112] GATCCAAAGTGGTACTTGTTTTTCCAAGGCCCAGGTCGTGGCATCACTA

[0113] TGAAAAACGGCACCATCGTATTCCCGGCCCAATTCAAAGATGAGAATC

[0114] AGGTTCCTCACTCGACTATTATCTACTCTAAAGATCACGGGAAGACGTG

[0115] GTCGGTTGGCACTGGTGCTAAGTCCAAAACGACGGAGGCCCAGGTTGT

[0116] TGAATTATCAGATGGGTCGTTAATGTTGAATATGCGGGACGATCGCGGT

[0117] GGCTTCCGCTCAATCGCAGTTACTAGTGATATGGGTAAGACATGGGTTG

[0118] AGCACTCTAGTTCCCGCTCGGCGCTTCCGGAAAGTACGTGTATGGGGA

[0119] GCATCATCACATTGAAAACAAAGAAAGGCGAGAACATTATGTTGTTTT

[0120] CCAATCCTAATACGCAAAAGGGCCGCCATTCAATGACCATTAAAGTCT

[0121] CGTTTGATGAAGGCAAGACCTGGCCTGTGGAAAATAATTTACTTTACCA

[0122] CGCGGGGGACTGTTTCGGTTATTCTTGTTTAACCCAGATTGATGATAAG

[0123] ACGGTCGGGGTATTGTATGAAGGCGTTCGTGATTTGTACTTCCAAAAAT

[0124] TTGCTATTGAAGAACTCTTACACCCTAAGATGAAAAAGGGGGGGAGTG

[0125] GCGGCTCTGGGGGCTCTGAGGGAGGCTCAGGTGGGTCGATGGGCGCGA

[0126] TGGTGGATACCCTGAGCGGCCTGAGCAGCGAACAGGGCCAGAGCGGCG

[0127] ATATGACCATTGAAGAAGATAGCGCGACCCATATTAAATTTAGCAAAC

[0128] GCGATGAAGATGGCAAAGAACTGGCGGGCGCGACCATGGAACTGCGC

[0129] GATAGCAGCGGCAAAACCATTAGCACCTGGATTAGCGATGGCCAGGTG

[0130] AAAGATTTTTATCTGTATCCGGGCAAATATACCTTTGTGGAAACCGCGG

[0131] CGCCGGATGGCTATGAAGTGGCGACCGCGATTACCTTTACCGTGAACG

[0132] AACAGGGCCAGGTGACCGTGAACGGCAAAGCGACCAAAGGCGATGCGCATATTTAA.

[0133]

[0134] The present invention also provides a host for transforming and / or transfecting the above-mentioned recombinant expression vector composition.

[0135] The present invention also provides a multi-enzyme complex, which is directly or indirectly prepared via the above-mentioned recombinant expression vector composition and / or the above-mentioned host.

[0136] In some embodiments of the present invention, in the above multi-enzyme complex, the amino acid sequence of the CSS is as shown in SEQ ID NO: 21: MKIIMTRIAIIPARAGSKGIKDKNLQLVGGVSLV GRAILAAQESGMFDQIVVTSDGENILKEATKYGAKPVARPESLAQSDTRTIDAILHCLETLNISQGTAALLQPTSPLRNALDIRNAMEIFLGGKYKSVVSACECEHHPYKSFTLEGTEVQPIHELTDFESPRQKLPKSYRANGAIYINDIQSLFEEKRFFIAPMRFYLMPTYRSIDIDSTLDLQLAESLISKEF;

[0137] The amino acid sequence of the CMK is shown in SEQ ID NO:22: MNIVPVITLDGPGGV GKGTLGRLLAHDLGWHLLDSGAIYRALALAAQRAGLDADNETALSGLARTLPLGFRGAADETHVLLGGEVVDAEIRTPEVSALTSRIAAIPAVRAALLQRQRDFRRAPGLVADGRDMGTVVFPDAPLKVFLTASAEERGKRRLNQLMEQGISASLATVVAEIAERDARDQQRSVAPLRPAPDAKILDTSEQSVSDTYRVLQSWVRSAFQEN;

[0138] The amino acid sequence of the PPK is shown in SEQ ID NO: 23: MARNGNGRKDAAGKAKSLPDLPFVGAITRYLEKDAPEALRKAVAAADGKRILDPSYPHAEEMKGKDYDDRMAGLQLQLVRLMRDVIHTGKRMVVLFEGRDAAGKGGTIERVRENLNPRSAYIVALPKPSEREAEEWYFQRYVDWLPAKGEIALFDRSWYNRGVVEKVFGFCTDAQRETFFRQLPLFENMLVDDGVILVKLWLEVGRAEQLKRFLDREQDPLKQWKLSPIDVEGLAKWEDYSAAIQDTLLRSHSGIAPWTVIRADDKRRARLAAIQTILRAVDFAGRDDEVIGQPDPQITGGPEILPG;

[0139] The amino acid sequence of the 3-ST is shown in SEQ ID NO: 24: MKTITLYLDPASLPA LNQLMDFTQNNEDKTHPRIFGLSRFKIPDNIITQYQNIHFVELKDNRPTEALFTILDQYPGNIELDIHLNIAHSVQLIRPILAYRFKHLDRVSIQRLNLYDDGSMEYVDLEKEENKDISAEIKQAEKQLSHYLLTGKIKFDNPTIARYVWQSAFPVKYHFLSTDYFEKAEFLQPLKEYLAENYQKMDWTAYQQLTPEQQAFYLTLVGFNDEVKQSLEVQQAKFIFTGTTTWEGNTDVREYYAQQQLNLLNHFTQAGGDLFIGDHYKIYFKGHPRGGEINDYILNNAKNITNIPANISFEVLMMTGLLPDKVGGVASSLYFSLPKEKISHIIFTSNKQVKSKEDALNNPYVKVMRRLGIIDESQVIFWDSLKQL;

[0140] The amino acid sequence of 3-ST is shown in SEQ ID NO:25: MRILFVVAFFASLAA VCQAQSSLQAEVLQKQNPVLVGVKNNVALCLKVSVPENQQIAISELRFSTKGTSRLSDLKAFRVFSTGTRSSFVDSAMVSQTAKVGKELAVKEAIMLQKGENYLWVTLELKDKVNLLNRFDVACSS MTLSNGAVLTPTDRTPNVVLRAGYAVRQRMQDGVNTYRIPGLVTTKKGTLISVFDIRRKYSGDLQGDIDIGICRSMDGGKSWLPMQVAIDMGEWGGLSHDENGITDPSILVDETTGTVWIAALWLNG SKGKPAWWASKPGMTPDVTGQFVLAKSDDDGLTWSEPINVTEQMKDPKWYLFFQGPGRGITMKNGTIVFPAQFKDENQVPHSTIIYSKDHGKTWSVGTGAKSKTTEAQVVELSDGSLMLNMRDDRG GFRSIAVTSDMGKTWVEHSSSRSALPESTCMGSIITLKTKKGENIMLFSNPNTQKGRHSMTIKVSFDEGKTWPVENNLLYHAGDCFGYSCLTQIDDKTVGVLYEGVRDLYFQKFAIEELLHPKMKK.

[0141] In some embodiments of the present invention, the above-mentioned multi-enzyme complex includes a CSS complex, a CMP-PPK complex and a ST complex;

[0142] The CSS complex includes:

[0143] SpyTag-CSS-SnoopTag, whose amino acid sequence is shown in SEQ ID NO:26: MAHIV MVDAYKPTKGGGGSGGGGSGGGGSMKIIMTRIAIIPARAGSKGIKDKNLQLVGGVSLVGRAILAAQESGMFDQIVVTSDGENILKEATKYGAKPVARPESLAQSDTRTIDAILHCLETLNISQGTAALLQPTSPLRNALDIRNAMEIFLGGKYKSVVSACECEHHPYKSFTLEGTEVQPIHELTDFESPRQKLPKSYRANGAIYINDIQSLFEEKRFFIAPMRFYLMPTYRSIDIDSTLDLQLAESLISKEFGGSGGSGGSEGGSGGSMKLGDIEFIKVNK;

[0144] SpyTag-CSS-RIAD, whose amino acid sequence is shown in SEQ ID NO:27: MAHIVMV DAYKPTKGGGGSGGGGSGGGGSMKIIMTRIAIIPARAGSKGIKDKNLQLVGGVSLVGRAILAAQESGMFDQIVVTSDGENILKEATKYGAKPVARPESLAQSDTRTIDAILHCLETLNISQGTAALLQPTSPLRNALDIRNAMEIFLGGKYKSVVSACECEHHPYKSFTLEGTEVQPIHELTDFESPRQKLPKSYRANGAIYINDIQSLFEEKRFFIAPMRFYLMPTYRSIDIDSTLDLQLAESLISKEFGGSGGSGGSEGGSGGSMLEQYANQLADQIIKEATE;

[0145] RIAD-CSS-SdyTag, whose amino acid sequence is shown in SEQ ID NO:28: MLEQYAN QLADQIIKEATEGGGGSGGGGSGGGGSMKIIMTRIAIIPARAGSKGIKDKNLQLVGGVSLVGRAILAAQESGMFDQIVVTSDGENILKEATKYGAKPVARPESLAQSDTRTIDAILHCLETLNISQGTAALLQPTSPLRNALDIRNAMEIFLGGKYKSVVSACECEHHPYKSFTLEGTEVQPIHELTDFESPRQKLPKSYRANGAIYINDIQSLFEEKRFFIAPMRFYLMPTYRSIDIDSTLDLQLAESLISKEFGGSGGSGGSEGGSGGSMDPIVMIDNDKPIT;

[0146] SpyTag-CSS-SdyTag, whose amino acid sequence is shown in SEQ ID NO:29: MAHIVMV DAYKPTKGGGGSGGGGSGGGGSMKIIMTRIAIIPARAGSKGIKDKNLQLVGGVSLVGRAILAAQESGMFDQIVVTSDGENILKEATKYGAKPVARPESLAQSDTRTIDAILHCLETLNISQGTAALLQPTSPLRNALDIRNAMEIFLGGKYKSVVSACECEHHPYKSFTLEGTEVQPIHELTDFESPRQKLPKSYRANGAIYINDIQSLFEEKRFFIAPMRFYLMPTYRSIDIDSTLDLQLAESLISKEFGGSGGSGGSEGGSGGSMDPIVMIDNDKPIT;

[0147] SnoopTag-CSS-SpyTag, whose amino acid sequence is shown in SEQ ID NO:30: MHMKP LRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIVPQDIPATYEFTNGKHYITNEPIPPKGGSGGSGGSEGGSGGSMKIIMTRIAIIPARAGSKGIKDKNLQLVGGVSLVGRAILAAQESGMFDQIVVTSDGENILKEATKYGAKPVARPESLAQSDTRTIDAILHCLETLNISQGTAALLQPTSPLRNALDIRNAMEIFLGGKYKSVVSACECEHHPYKSFTLEGTEVQPIHELTDFESPRQKLPKSYRANGAIYINDIQSLFEEKRFFIAPMRFYLMPTYRSIDIDSTLDLQLAESLISKEFGGGGSEAAKEAAKGGGGSMAHIVMVDAYKPTK;

[0148] The CMK-PPK complex includes:

[0149] CMK-PPK-SpyCatcher, whose amino acid sequence is shown in SEQ ID NO: 31: MNIVPV ITLDGPGGVGKGTLGRLLAHDLGWHLLDSGAIYRALALAAQRAGLDADNETALSGLARTLPLGFRGAADETHVLLGGEVVDAEIRTPEVSALTSRIAAIPAVRAALLQRQRDFRRAPGLVADGRDMGTVVFPDAPLKVFLTASAEERGKRRLNQLMEQGISASLATVVAEIAERDARDQQRSVAPLRPAPDAKILDTSEQSVSDTYRVLQSWVRSAFQENGGSGGSGGSEGGSGGSMARNGNGRKDAAGKAKSLPDLPFVGAITRYLEKDAPEALRKAVAAADGKRILDPSYPHAEEMKGKDYDDRMAGLQLQLVRLMRDVIHTGKRMVVLFEGRDAAGKGGTIERVRENLNPRSAYIVALPKPSEREAEEWYFQRYVDWLPAKGEIALFDRSWYNRGVVEKVFGFCTDAQRETFFRQLPLFENMLVDDGVILVKLWLEVGRAEQLKRFLDREQDPLKQWKLSPIDVEGLAKWEDYSAAIQDTLLRSHSGIAPWTVIRADDKRRARLAAIQTILRAVDFAGRDDEVIGQPDPQITGGPEILPGGGGGSEAAKEAAKGGGGSMGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI;

[0150] CMK-PPK-RIDD, whose amino acid sequence is shown in SEQ ID NO:32: MNIVPVITLD GPGGVGKGTLGRLLAHDLGWHLLDSGAIYRALALAAQRAGLDADNETALSGLARTLPLGFRGAADETHVLLGGEVVDAEIRTPEVSALTSRIAAIPAVRAALLQRQRDFRRAPGLVADGRDMGTVVFPDAPLKVFLTASAEERGKRRLNQLMEQGISASLATVVAEIAERDARDQQRSVAPLRPAPDAKILDTSEQSVSDTYRVLQSWVRSAFQENGGSGGSGGSEGGSGGSMARNGNGRKDAAGKAKSLPDLPFVGAITRYLEKDAPEALRKAVAAADGKRILDPSYPHAEEMKGKDYDDRMAGLQLQLVRLMRDVIHTGKRMVVLFEGRDAAGKGGTIERVRENLNPRSAYIVALPKPSEREAEEWYFQRYVDWLPAKGEIALFDRSWYNRGVVEKVFGFCTDAQRETFFRQLPLFENMLVDDGVILVKLWLEVGRAEQLKRFLDREQDPLKQWKLSPIDVEGLAKWEDYSAAIQDTLLRSHSGIAPWTVIRADDKRRARLAAIQTILRAVDFAGRDDEVIGQPDPQITGGPEILPGGGGGSEAAKEAAKGGGGSMLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK;

[0151] CMK-PPK-SdyCatcher, whose amino acid sequence is shown in SEQ ID NO:33: MNIVPV ITLDGPGGVGKGTLGRLLAHDLGWHLLDSGAIYRALALAAQRAGLDADNETALSGLARTLPLGFRGAADETHVLLGGEVVDAEIRTPEVSALTSRIAAIPAVRAALLQRQRDFRRAPGLVADGRDMGTVVFPDAPLKVFLTASAEERGKRRLNQLMEQGISASLATVVAEIAERDARDQQRSVAPLRPAPDAKILDTSEQSVSDTYRVLQSWVRSAFQENGGSGGSGGSEGGSGGSMARNGNGRKDAAGKAKSLPDLPFVGAITRYLEKDAPEALRKAVAAADGKRILDPSYPHAEEMKGKDYDDRMAGLQLQLVRLMRDVIHTGKRMVVLFEGRDAAGKGGTIERVRENLNPRSAYIVALPKPSEREAEEWYFQRYVDWLPAKGEIALFDRSWYNRGVVEKVFGFCTDAQRETFFRQLPLFENMLVDDGVILVKLWLEVGRAEQLKRFLDREQDPLKQWKLSPIDVEGLAKWEDYSAAIQDTLLRSHSGIAPWTVIRADDKRRARLAAIQTILRAVDFAGRDDEVIGQPDPQITGGPEILPGGGGGSEAAKEAAKGGGGSMGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSSGLSGETGQSGNTTIEEDSTTHVKFSKRDANGKELAGAMIELRNLSGQTIQSWISDGTVKVFYLMPGTYQFVETAAPEGYELAAPITFTIDEKGQIWVDS;

[0152] SpyCatcher-CMK-PPK, whose amino acid sequence is shown in SEQ ID NO:34: MGAMV DTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGGGSGGGGSGGGGSMNIVPVITLDGPGGVGKGTLGRLLAHDLGWHLLDSGAIYRALALAAQRAGLDADNETALSGLARTLPLGFRGAADETHVLLGGEVVDAEIRTPEVSALTSRIAAIPAVRAALLQRQRDFRRAPGLVADGRDMGTVVFPDAPLKVFLTASAEERGKRRLNQLMEQGISASLATVVAEIAERDARDQQRSVAPLRPAPDAKILDTSEQSVSDTYRVLQSWVRSAFQENGGSGGSGGSEGGSGGSMARNGNGRKDAAGKAKSLPDLPFVGAITRYLEKDAPEALRKAVAAADGKRILDPSYPHAEEMKGKDYDDRMAGLQLQLVRLMRDVIHTGKRMVVLFEGRDAAGKGGTIERVRENLNPRSAYIVALPKPSEREAEEWYFQRYVDWLPAKGEIALFDRSWYNRGVVEKVFGFCTDAQRETFFRQLPLFENMLVDDGVILVKLWLEVGRAEQLKRFLDREQDPLKQWKLSPIDVEGLAKWEDYSAAIQDTLLRSHSGIAPWTVIRADDKRRARLAAIQTILRAVDFAGRDDEVIGQPDPQITGGPEILPG;

[0153] The ST complex includes:

[0154] 3-ST-SnoopCatcher, whose amino acid sequence is shown in SEQ ID NO:35: MKTITLYL DPASLPALNQLMDFTQNNEDKTHPRIFGLSRFKIPDNIITQYQNIHFVELKDNRPTEALFTILDQYPGNIELDIHLNIAHSVQLIRPILAYRFKHLDRVSIQRLNLYDDGSMEYVDLEKEENKDISAEIKQAEKQLSHYLLTGKIKFDNPTIARYVWQSAFPVKYHFLSTDYFEKAEFLQPLKEYLAENYQKMDWTAYQQLTPEQQAFYLTLVGFNDEVKQSLEVQQAKFIFTGTTTWEGNTDVREYYAQQQLNLLNHFTQAGGDLFIGDHYKIYFKGHPRGGEINDYILNNAKNITNIPANISFEVLMMTGLLPDKVGGVASSLYFSLPKEKISHIIFTSNKQVKSKEDALNNPYVKVMRRLGIIDESQVIFWDSLKQLGGGGSEAAKEAAKGGGGSMHMKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQNVRTGEDGKLTFKNLSDGKYRLFENSEPAGYKPVQNKPIVAFQIVNGEVRDVTSIVPQDIPATYEFTNGKHYITNEPIPPK;

[0155] 3-ST-SpyCatcher, whose amino acid sequence is shown in SEQ ID NO:36: MKTITLYLDP ASLPALNQLMDFTQNNEDKTHPRIFGLSRFKIPDNIITQYQNIHFVELKDNRPTEALFTILDQYPGNIELDIHLNIAHSVQLIRPILAYRFKHLDRVSIQRLNLYDDGSMEYVDLEKEENKDISAEIKQAEKQLSHYLLTGKIKFDNPTIARYVWQSAFPVKYHFLSTDYFEKAEFLQPLKEYLAENYQKMDWTAYQQLTPEQQAFYLTLVGFNDEVKQSLEVQQAKFIFTGTTTWEGNTDVREYYAQQQLNLLNHFTQAGGDLFIGDHYKIYFKGHPRGGEINDYILNNAKNITNIPANISFEVLMMTGLLPDKVGGVASSLYFSLPKEKISHIIFTSNKQVKSKEDALNNPYVKVMRRLGIIDESQVIFWDSLKQLGGGGSGGGGSGGGGSMGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI;

[0156] SdyCatcher-3-ST, whose amino acid sequence is shown in SEQ ID NO:37: MGSSHHHHH HSSGLVPRGSHMASMTGGQQMGRGSSGLSGETGQSGNTTIEEDSTTHVKFSKRDANGKELAGAMIELRNLSGQTIQSWISDGTVKVFYLMPGTYQFVETAAPEGYELAAPITFTIDEKGQIWVDSGGGGSGGGGSGGGGSMKTITLYLDPASLPALNQLMDFTQNNEDKTHPRIFGLSRFKIPDNIITQYQNIHFVELKDNRPTEALFTILDQYPGNIELDIHLNIAHSVQLIRPILAYRFKHLDRVSIQRLNLYDDGSMEYVDLEKEENKDISAEIKQAEKQLSHYLLTGKIKFDNPTIARYVWQSAFPVKYHFLSTDYFEKAEFLQPLKEYLAENYQKMDWTAYQQLTPEQQAFYLTLVGFNDEVKQSLEVQQAKFIFTGTTTWEGNTDVREYYAQQQLNLLNHFTQAGGDLFIGDHYKIYFKGHPRGGEINDYILNNAKNITNIPANISFEVLMMTGLLPDKVGGVASSLYFSLPKEKISHIIFTSNKQVKSKEDALNNPYVKVMRRLGIIDESQVIFWDSLKQL;

[0157] SdyCatcher-6-ST, whose amino acid sequence is shown in SEQ ID NO:38: MGSSHHHHH HSSGLVPRGSHMASMTGGQQMGRGSSGLSGETGQSGNTTIEEDSTTHVKFSKRDANGKELAGAMIELRNLSGQTIQSWISDGTVKVFYLMPGTYQFVETAAPEGYELAAPITFTIDEKGQIWVDSGGGGSGGGGSGGGGSMRILFVVAFFASLAAVCQAQSSLQAEVLQKQNPVLVGVKNNVALCLKVSVPENQQIAISELRFSTKGTSRLSDLKAFRVFSTGTRSSFVDSAMVSQTAKVGKELAVKEAIMLQKGENYLWVTLELKDKVNLLNRFDVACSSMTLSNGAVLTPTDRTPNVVLRAGYAVRQRMQDGVNTYRIPGLVTTKKGTLISVFDIRRKYSGDLQGDIDIGICRSMDGGKSWLPMQVAIDMGEWGGLSHDENGITDPSILVDETTGTVWIAALWLNGSKGKPAWWASKPGMTPDVTGQFVLAKSDDDGLTWSEPINVTEQMKDPKWYLFFQGPGRGITMKNGTIVFPAQFKDENQVPHSTIIYSKDHGKTWSVGTGAKSKTTEAQVVELSDGSLMLNMRDDRGGFRSIAVTSDMGKTWVEHSSSRSALPESTCMGSIITLKTKKGENIMLFSNPNTQKGRHSMTIKVSFDEGKTWPVENNLLYHAGDCFGYSCLTQIDDKTVGVLYEGVRDLYFQKFAIEELLHPKMKK;

[0158] 6-ST-SpyCatcher, whose amino acid sequence is shown in SEQ ID NO:39: MRILFVVAFF ASLAAVCQAQSSLQAEVLQKQNPVLVGVKNNVALCLKVSVPENQQIAISELRFSTKGTSRLSDLKAFRVFSTGTRSSFVDSAMVSQTAKVGKELAVKEAIMLQKGENYLWVTLELKDKVNLLNRFDVACSSMTLSNGAVLTPTDRTPNVVLRAGYAVRQRMQDGVNTYRIPGLVTTKKGTLISVFDIRRKYSGDLQGDIDIGICRSMDGGKSWLPMQVAIDMGEWGGLSHDENGITDPSILVDETTGTVWIAALWLNGSKGKPAWWASKPGMTPDVTGQFVLAKSDDDGLTWSEPINVTEQMKDPKWYLFFQGPGRGITMKNGTIVFPAQFKDENQVPHSTIIYSKDHGKTWSVGTGAKSKTTEAQVVELSDGSLMLNMRDDRGGFRSIAVTSDMGKTWVEHSSSRSALPESTCMGSIITLKTKKGENIMLFSNPNTQKGRHSMTIKVSFDEGKTWPVENNLLYHAGDCFGYSCLTQIDDKTVGVLYEGVRDLYFQKFAIEELLHPKMKKGGSGGSGGSEGGSGGSMGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI;

[0159] 6-ST-SnoopCatcher, the amino acid sequence of which is shown in SEQ ID NO:40:.

[0160] The present invention also provides a method for preparing the multi-enzyme complex, comprising the following steps:

[0161] S1: obtaining the above-mentioned recombinant expression vector composition;

[0162] S2: Take the recombinant expression vector composition, transform and express it separately to obtain the multi-enzyme complex.

[0163] The present invention also provides a method for preparing sialyllactose, which is obtained by using sialic acid as a starting material and catalyzed by the multi-enzyme complex or the multi-enzyme complex obtained by the preparation method.

[0164] The present invention also provides the use of the above recombinant expression vector composition, the above host, the above multi-enzyme complex, and the multi-enzyme complex obtained by the above preparation method in the preparation of sialyllactose and / or products containing sialyllactose.

[0165] The present invention provides an application of a protein group in the preparation of sialyllactose; the protein group includes a first protein and a second protein; the first protein includes any two of SpyTag, SnoopTag, SdyTag or RIAD; the second protein includes any two of SpyCatcher, SnoopCatcher, SdyCatcher or RIDD; the first protein and the second protein are covalently bound to form a protein interaction pair. The present invention also provides a recombinant expression vector composition, a multi-enzyme complex and an application in the preparation of sialyllactose.

[0166] This patent realizes the orderly assembly of multiple enzymes in two-dimensional space through two orthogonal protein interaction pairs (such as SpyTag / SpyCatcher, SnoopTag / SnoopCatcher, SdyTag / SdyCatcher and RIAD / RIDD), achieving efficient preparation of sialyllactose (short time, high yield, high product concentration). Considering that an effective CTP regeneration system is an important factor affecting the yield of sialyllactose, this patent fusion-expresses CMP kinase (CMK) and polyphosphate kinase (PPK), reducing the amount of enzymes required for the process while ensuring sufficient CTP supply. BRIEF DESCRIPTION OF THE DRAWINGS

[0167] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0168] Figure 1 Demonstrate the technical route of this patent;

[0169] Figure 2 The HPLC spectrum of 3'-SL prepared in Example 1 is shown; wherein: from top to bottom are 3'-SL standard product, reaction 0h, and reaction end point;

[0170] Figure 3 The HPLC spectrum of 3'-SL prepared in Example 2 is shown; wherein: from top to bottom are 3'-SL standard product, reaction 0h, and reaction end point;

[0171] Figure 4 The HPLC spectrum of 3'-SL prepared in Example 3 is shown; wherein: from top to bottom are 3'-SL standard product, reaction 0h, and reaction end point;

[0172] Figure 5The HPLC spectrum of 6'-SL prepared in Example 4 is shown; wherein: from top to bottom are 6'-SL standard product, reaction 0h, and reaction end point;

[0173] Figure 6 The HPLC spectrum of 6'-SL prepared in Example 5 is shown; wherein: from top to bottom are 6'-SL standard product, reaction 0h, and reaction end point;

[0174] Figure 7 The HPLC spectrum of 6'-SL prepared in Example 6 is shown; wherein: from top to bottom are 6'-SL standard product, reaction 0h, and reaction end point;

[0175] Figure 8 The HPLC spectrum of 3'-SL prepared in Comparative Example 1 is shown; wherein: from top to bottom are 3'-SL standard product, reaction 0h, and reaction end point;

[0176] Fig. 9 The HPLC spectrum of 6'-SL prepared in Comparative Example 2 is shown; wherein: from top to bottom are 6'-SL standard product, reaction 0h, and reaction end point. DETAILED DESCRIPTION

[0177] The invention discloses a protein group, a multi-enzyme complex and application thereof and a preparation method of sialyllactose.

[0178] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0179] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0180] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0181] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0182] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0183] In Examples 1 to 6, Comparative Example 1, Comparative Example 2 and Effect Example 1 of the present invention,

[0184] Chinese-English comparison table:

[0185]

[0186]

[0187] The source of the enzyme involved in the present invention is:

[0188] CMP-SA synthetase was derived from Haemophilus influenzae ATCC 51907 (Uniprot #Q57140).

[0189] 2,3-Sialyllactosyltransferase is derived from Pasteurella multocida by cleaving off the N-terminal 24 amino acids (Uniprot#Q9CP67).

[0190] 2,6-Sialyl lactosyltransferase was derived from Acetobacteroides hydrogenigenes (Uniprot#A0A4V2RQV8).

[0191] CMP kinase is derived from Acidithiobacillus ferrooxidans (Uniprot #B7J782).

[0192] Polyphosphate kinase was derived from Paracoccus sp. (Uniprot #A0A0V0Q0V3).

[0193] All the raw materials and reagents used can be purchased from the market.

[0194] The present invention will be further described below in conjunction with embodiments:

[0195] Example 1 Preparation of 3'-SL by Multienzyme Complex 1

[0196] SpyTag and SnoopTag were connected to the N-terminus and C-terminus of CSS respectively, and the fusion fragments were inserted into the pET-28a vector to obtain the pET28a-SpyTag-CSS-SnoopTag recombinant plasmid; the CMK gene and PPK gene were fused with the SpyCatcher gene and inserted into the pRSFDuet vector to obtain the pRSFDuet-CMK-PPK-SpyCatcher recombinant plasmid; SnoopCatcher was connected to the C-terminus of 3-ST and inserted into the pET-28a vector to obtain the pET28a-3-ST-SnoopCatcher recombinant plasmid; the recombinant plasmid with confirmed correct sequence was transferred into E. coli (BL21) competent cells for plate culture, and a single clone was picked for small-scale liquid culture. The bacteria with correct protein expression were finally amplified step by step for liquid culture, and the bacteria after large-scale expression were crushed and centrifuged to remove the cell wall to obtain the corresponding crude enzyme solution.

[0197] 1L reaction system contains 200mM sialic acid, 5mM CMP, 250mM lactose, 400mM magnesium chloride, and 100mM sodium polyphosphate. 1000U SpyTag-CSS-SnoopTag, 1500U CMK-PPK-SpyCatcher, and 800U 3-ST-SnoopCatcher are added to the reaction solution. The reaction is stirred at 30°C, and the pH is controlled to 7.5 with sodium hydroxide. After the reaction is carried out for 5h, a small amount of the reaction solution is centrifuged at 12000rpm for 2min, and the supernatant is filtered for HPLC detection (such as Figure 2 As shown). After testing, the 3'-SL content was 110.2 g / L (conversion rate 87%).

[0198] Example 2 Preparation of 3'-SL by Multienzyme Complex 2

[0199] SpyTag and RIAD were connected to the N-terminus and C-terminus of CSS respectively, and the fusion fragments were inserted into the pET-28a vector to obtain the pET28a-SpyTag-CSS-RIAD recombinant plasmid; the CMK gene, PPK gene and RIDD gene were fused and inserted into the pRSFDuet vector to obtain the pRSFDuet-CMK-PPK-RIDD recombinant plasmid; SpyCatcher was connected to the C-terminus of 3-ST and inserted into the pET-28a vector to obtain the pET28a-3-ST-SpyCatcher recombinant plasmid; the recombinant plasmid with confirmed correct sequence was transferred into E. coli (BL21) competent cells for plate culture, and a single clone was picked for small-scale liquid culture. The bacteria with correct protein expression were finally amplified step by step for liquid culture, and the bacteria after large-scale expression were crushed and centrifuged to remove the cell wall to obtain the corresponding crude enzyme solution.

[0200] 1L reaction system contains 200mM sialic acid, 10mM CMP, 220mM lactose, 500mM magnesium chloride, and 120mM sodium polyphosphate. 800U SpyTag-CSS-RIAD, 1000U CMK-PPK-RIDD, and 1000U 3-ST-SpyCatcher are added to the reaction solution. The reaction is stirred at 30°C, and the pH is controlled to 7.5 with sodium hydroxide. The reaction is continued for 4h. A small amount of the reaction solution is centrifuged at 12000rpm for 2min, and the supernatant is filtered for HPLC detection (such as Figure 3 As shown). After testing, the 3'-SL content was 120.4 g / L (conversion rate 95%).

[0201] Example 3 Preparation of 3'-SL by Multienzyme Complex 3

[0202] SpyTag and SdyTag were connected to the N-terminus and C-terminus of CSS respectively, and the fusion fragments were inserted into the p ET-28a vector to obtain the pET28a-SpyTag-CSS-SdyTag recombinant plasmid; the CMK gene and PPK gene were fused with the SpyCatcher gene and inserted into the pRSFDuet vector to obtain the pRSFDuet-SpyCatcher-CMK-PPK recombinant plasmid; SdyCatcher was connected to the N-terminus of 3-ST and inserted into the pET-28a vector to obtain the pET28a-SdyCatcher-3-ST recombinant plasmid; the recombinant plasmid with confirmed correct sequence was transferred into E. coli (BL21) competent cells for plate culture, and a single clone was picked for small-scale liquid culture. The bacteria with correct protein expression were finally amplified step by step for liquid culture, and the bacteria after large-scale expression were crushed and centrifuged to remove the cell wall to obtain the corresponding crude enzyme solution.

[0203] 1L reaction system contains 200mM sialic acid, 15mM CMP, 360mM lactose, 700mM magnesium chloride, and 240mM sodium polyphosphate. 1500U SpyTag-CSS-SdyTag, 1200U SpyCatcher-CMK-PPK, and 1500U SdyCatcher-3-ST are added to the reaction solution. The reaction is stirred at 30°C, and the pH is controlled to 7.5 with sodium hydroxide. The reaction is continued for 3h. A small amount of the reaction solution is centrifuged at 12000rpm for 2min, and the supernatant is filtered for HPLC detection (such as Figure 4 As shown). After testing, the 3'-SL content was 119.2 g / L (conversion rate 94%).

[0204] Example 4 Multienzyme Complex 4 Preparation of 6'-SL

[0205] RIAD and SdyTag were connected to the N-terminus and C-terminus of CSS respectively, and the fusion fragments were inserted into the pET-28a vector to obtain the pET28a-RIAD-CSS-SdyTag recombinant plasmid; the CMK gene, PPK gene and RIDD gene were fused and inserted into the pRSFDuet vector to obtain the pRSFDuet-CMK-PPK-RIDD recombinant plasmid; SdyCatcher was connected to the N-terminus of 6-ST and inserted into the pET-28a vector to obtain the pET28a-SdyCatcher-6-ST recombinant plasmid; the recombinant plasmid with confirmed correct sequence was transferred into E. coli (BL21) competent cells for plate culture, and a single clone was picked for small-scale liquid culture. The bacteria with correct protein expression were finally amplified step by step for liquid culture, and the bacteria after large-scale expression were crushed and centrifuged to remove the cell wall to obtain the corresponding crude enzyme solution.

[0206] 1L reaction system contains 200mM sialic acid, 8mM CMP, 300mM lactose, 700mM magnesium chloride, and 200mM sodium polyphosphate. 1500U RIAD-CSS-SdyTag, 1000U CMK-PPK-RIDD, and 1200U SdyCatcher-6-ST are added to the reaction solution. The reaction is stirred at 30°C, and the pH is controlled to 7.5 with sodium hydroxide. The reaction is continued for 3h. A small amount of the reaction solution is centrifuged at 12000rpm for 2min, and the supernatant is filtered for HPLC detection (such as Figure 5 As shown). After testing, the 6'-SL content was 114.1 g / L (conversion rate 90%).

[0207] Example 5 Multienzyme Complex 5 Preparation of 6'-SL

[0208] SpyTag and SdyTag were connected to the N-terminus and C-terminus of CSS respectively, and the fusion fragments were inserted into the p ET-28a vector to obtain the pET28a-SpyTag-CSS-SdyTag recombinant plasmid; the CMK gene, PPK gene and SdyCatcher gene were fused and inserted into the pRSFDuet vector to obtain the pRSFDuet-CMK-PPK-SdyCatcher recombinant plasmid; SpyCatcher was connected to the C-terminus of 6-ST and inserted into the pET-28a vector to obtain the pET28a-6-ST-SpyCatcher recombinant plasmid; the recombinant plasmid with confirmed correct sequence was transferred into E. coli (BL21) competent cells for plate culture, and a single clone was picked for small-scale liquid culture. The bacteria with correct protein expression were finally amplified step by step for liquid culture, and the bacteria after large-scale expression were crushed and centrifuged to remove the cell wall to obtain the corresponding crude enzyme solution.

[0209] 1L reaction system contains 200mM sialic acid, 15mM CMP, 360mM lactose, 600mM magnesium chloride, and 240mM sodium polyphosphate. 800U SpyTag-CSS-SdyTag, 1000U CMK-PPK-SdyCatcher, and 1500U 6-ST-SpyCatcher are added to the reaction solution. The reaction is stirred at 30°C, and the pH is controlled to 7.5 with sodium hydroxide. After the reaction is carried out for 5h, a small amount of the reaction solution is centrifuged at 12000rpm for 2min, and the supernatant is filtered for HPLC detection (such as Figure 6 As shown). After testing, the 6'-SL content was 116.6 g / L (conversion rate 92%).

[0210] Example 6 Multienzyme Complex 6 Preparation of 6'-SL

[0211] SnoopTag and SpyTag were connected to the N-terminus and C-terminus of CSS respectively, and the fusion fragments were inserted into the pET-28a vector to obtain the pET28a-SnoopTag-CSS-SpyTag recombinant plasmid; the CMK gene and the PPK gene were fused with the SpyCatcher gene and inserted into the pRSFDuet vector to obtain the pRSFDuet-CMK-PPK-SpyCatcher recombinant plasmid; SnoopyCatcher was connected to the C-terminus of 6-ST and inserted into the pET-28a vector to obtain the pET28a-6-ST-SnoopCatcher recombinant plasmid; the recombinant plasmid with confirmed correct sequence was transferred into E. coli (BL21) competent cells for plate culture, and a single clone was picked for small-scale liquid culture. The bacteria with correct protein expression were finally amplified step by step for liquid culture, and the bacteria after large-scale expression were crushed and centrifuged to remove the cell wall to obtain the corresponding crude enzyme solution.

[0212] 1L reaction system contains 200mM sialic acid, 5mM CMP, 220mM lactose, 400mM magnesium chloride, and 100mM sodium polyphosphate. 1000U SnoopTag-CSS-Sp yTag, 1200U CMK-PPK-SpyCatcher, and 800U 6-ST-SnoopCatcher are added to the reaction solution. The reaction is stirred at 30°C, and the pH is controlled to 7.5 with sodium hydroxide. The reaction is continued for 4h. A small amount of the reaction solution is centrifuged at 12000rpm for 2min, and the supernatant is filtered for HPLC detection (such as Figure 7 As shown). After testing, the 6'-SL content was 115.4 g / L (conversion rate 91%).

[0213] Comparative Example 1 Preparation of 3'-SL by multi-enzyme catalysis (without assembly)

[0214] 1L reaction system contains 200mM sialic acid, 5mM CMP, 250mM lactose, 400mM magnesium chloride, 100mM sodium polyphosphate. 1000U CSS, 1500U CMK, 1500UPPK, 800U 3-ST are added to the reaction solution. The reaction is stirred at 30°C. The pH is controlled to 7.5 with sodium hydroxide. The reaction is continued for 10h. A small amount of the reaction solution is centrifuged at 12000rpm for 2min. The supernatant is filtered and tested by HPLC (such as Figure 8 As shown). After testing, the 3'-SL content was 71.0 g / L (conversion rate 56%).

[0215] Comparative Example 1 to 3, in the absence of assembly, the multi-enzyme catalysis was used to prepare 3'-SL, the conversion rate decreased by 30%, and the reaction time was doubled, which shows that the multi-enzyme complex catalyzed the production of 3'-SL with higher efficiency.

[0216] 1L reaction system contains 200mM sialic acid, 15mM CMP, 360mM lactose, 600mM magnesium chloride, and 240mM sodium polyphosphate. 800U CSS, 1000U CMK, 1000U PPK, and 1500U 6-ST are added to the reaction solution. The reaction is stirred at 30°C, and the pH is controlled to 7.5 with sodium hydroxide. The reaction is continued for 12h. A small amount of the reaction solution is centrifuged at 12000rpm for 2min, and the supernatant is filtered for HPLC detection (such as Fig. 9 As shown). After testing, the 6'-SL content was 77.2 g / L (conversion rate 61%).

[0217] Comparative Examples 4 to 6, when 6'-SL was prepared by multi-enzyme catalysis without assembly, the conversion rate decreased by 30% and the reaction time was doubled. This shows that the multi-enzyme complex is more efficient in catalyzing the production of 6'-SL.

[0218] Effect Example 1

[0219] The reaction data of the embodiments and comparative examples are shown in Table 1;

[0220] Table 1

[0221]

[0222]

[0223] Note: / indicates not detected. The contents of SA, CMP-SA, 3'-SL and 6'-SL were calculated according to the dilution multiples of the samples.

[0224] It can be seen from Table 1 that compared with the comparative example, the conversion rate of the embodiment of the present invention is increased by 30%, and the reaction time is shortened by half.

[0225] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of a protein group in the preparation of sialyllactose; the protein group comprises a first protein and a second protein; The first protein includes any two of SpyTag, SnoopTag, SdyTag or RIAD; The second protein includes any two of SpyCatcher, SnoopCatcher, SdyCatcher or RIDD; The first protein and the second protein are covalently bound to form a protein interaction pair.

2. The use according to claim 1, characterized in that The protein interaction pairs include: Spy Tag / SpyCatcher, SnoopTag / SnoopCatcher, SdyTag / SdyCatcher or RIAD / RIDD.

3. A recombinant expression vector composition, characterized in that: include: CSS recombinant expression vector, CMK-PPK recombinant expression vector and ST recombinant expression vector; The CSS recombinant expression vector is obtained by coupling CSS with the first protein-1 and the first protein-2 respectively; The CMK-PPK recombinant expression vector is obtained by fusing CMK and PPK and coupling with the second protein-1; The ST recombinant expression vector is obtained by coupling ST with a second protein-2; The first protein-1 and the first protein-2 are independently selected from the first protein and / or the second protein in the use according to claim 1; The first protein-1 and the first protein-2 are not identical; The second protein-1 and the second protein-2 are not identical; The second protein-1 is covalently bound to the first protein-1 to form the protein interaction pair in the use as claimed in claim 1; The second protein-2 is covalently bound to the first protein-2 to form the protein interaction pair in the use as claimed in claim 1.

4. The recombinant expression vector composition according to claim 3, characterized in that: The ST includes: 3-ST or 6-ST.

5. The recombinant expression vector composition according to claim 4, characterized in that: The CSS recombinant expression vector comprises: one or more of SpyTag-CSS-SnoopTag plasmid, SpyTag-CSS-RIAD plasmid, RIAD-CSS-SdyTag plasmid, SpyTag-CSS-SdyTag plasmid and / or SnoopTag-CSS-SpyTag plasmid; The CMK-PPK recombinant expression vector comprises: one or more of a CMK-PPK-SpyCatcher plasmid, a CMK-PPK-RIDD plasmid, a CMK-PPK-SdyCatcher plasmid and / or a SpyCatcher-CMK-PPK plasmid; The ST recombinant expression vector comprises: one or more of 3-ST-SnoopCatcher plasmid, 3-ST-SpyCatcher plasmid, SdyCatcher-3-ST plasmid, SdyCatcher-6-ST plasmid, 6-ST-SpyCatcher plasmid and / or 6-ST-SnoopCatcher plasmid.

6. The recombinant expression vector composition according to any one of claims 3 to 5, characterized in that: The CSS has: (1), the nucleotide sequence shown in SEQ ID NO: 1; or (2) a nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (1), and having the same or similar function as the nucleotide sequence shown in (1); or (3) a nucleotide sequence having at least 80% identity with the nucleotide sequence shown in (1) or (2); and The CMK has: (4) the nucleotide sequence shown in SEQ ID NO: 2; or (5) a nucleotide sequence obtained by substituting, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as the nucleotide sequence shown in (4); or (6) a nucleotide sequence having at least 80% identity with the nucleotide sequence shown in (4) or (5); and The PPK has: (7), the nucleotide sequence shown in SEQ ID NO: 3; or (8) a nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (7), and having the same or similar function as the nucleotide sequence shown in (7); or (9) a nucleotide sequence having at least 80% identity with the nucleotide sequence shown in (7) or (8); and The ST has: (10), the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5; or (11) a nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (10), and having the same or similar function as the nucleotide sequence shown in (10); or (12) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (10) or (11).

7. A host, characterized in that Transformation and / or transfection is carried out according to the recombinant expression vector composition described in any one of claims 3 to 6.

8. A multi-enzyme complex, characterized in that The method is directly or indirectly obtained by the recombinant expression vector composition according to any one of claims 3 to 6 and / or the host according to claim 7.

9. A method for preparing a multi-enzyme complex, characterized in that: The following steps are involved: S1: obtaining the recombinant expression vector composition according to any one of claims 3 to 6; S2: Take the recombinant expression vector composition, transform and express it separately to obtain the multi-enzyme complex.

10. A method for preparing sialyllactose, characterized in that: Using sialic acid as the starting material It is obtained by catalysis of the multi-enzyme complex as described in claim 8 or the multi-enzyme complex obtained by the preparation method as described in claim 9.

11. Use of the recombinant expression vector composition according to any one of claims 3 to 6, the host according to claim 7, the multi-enzyme complex according to claim 8, and the multi-enzyme complex obtained by the preparation method according to claim 9 in the preparation of sialyllactose and / or products containing sialyllactose.

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