Fusion protein and use thereof in preparation of metabolites

Abstract

The present application discloses a fusion protein and a use thereof in preparation of metabolites. The fusion protein comprises a localization peptide of a first subcellular structure and an ESCRT protein. For a host capable of expressing the fusion protein, the fusion protein can be located on the surface of the subcellular structure, the subcellular structure storing target metabolites enters a sorting link through an ESCRT path, finally enters an exosome and is secreted out of a cell, so that the extracellular yield of the target metabolites is effectively increased.

Description

Fusion proteins and their applications in preparing metabolites Technical Field

[0001] The present application relates to the field of bioengineering technology, and in particular to a fusion protein and its application in preparing metabolites. Background Art

[0002] Microorganisms produce a wide variety of metabolites during their metabolic processes. Based on the relationship between these metabolites and microbial growth and reproduction, these metabolites can be divided into two categories: primary metabolites and secondary metabolites. Primary metabolites are substances produced by microorganisms through metabolic activity and necessary for their growth and reproduction. Secondary metabolites are substances produced at a certain stage of microbial growth that are chemically complex, have no apparent physiological function for the microorganism, or are not essential for microbial growth and reproduction. Secondary metabolites include, but are not limited to, terpenes, alkaloids, and flavonoids. Technical issues

[0003] Secondary metabolites synthesized by microorganisms are typically found intracellularly. Extracting these secondary metabolites often requires cell disruption, separation, and purification, which are time-consuming and labor-intensive, resulting in high extraction costs and reduced product purity. Therefore, developing a protein that can promote the extracellular secretion of target secondary metabolites is crucial for simplifying and reducing the production process and costs of these products, and is therefore beneficial for promoting their application and development. Technical Solutions

[0004] In view of this, the present application provides a fusion protein and its application in the preparation of metabolites, so as to provide a protein that can promote the secretion of target secondary metabolites to the extracellular space and be used in the preparation of target secondary metabolites.

[0005] In a first aspect, the present application provides a fusion protein comprising a localization peptide of a first subcellular structure and an ESCRT protein, wherein the ESCRT protein comprises one or more of the following (a) to (f):

[0006] (a) one or more proteins in the ESCRT-0 complex;

[0007] (b) one or more proteins in the ESCRT-I complex;

[0008] (c) one or more proteins in the ESCRT-II complex;

[0009] (d) one or more proteins in the ESCRT-III complex;

[0010] (e) one or more proteins in the Vps4-Vta1 complex;

[0011] (f) One or more proteins in the ALIX dimer.

[0012] Optionally, the one or more proteins in the ESCRT-0 complex are selected from one or more of Vps27, Hse1, HRS, STAM1 and STAM2;

[0013] The one or more proteins in the ESCRT-I complex are selected from one or more of Vps23, Vps28, Vps37, Mvb12, STP22, TSG101, VPS28, VPS37A, VPS37B, VPS37C, VPS37D, MVB12A, MVB12B, UBAP1, UBA1L and UMAD1;

[0014] The one or more proteins in the ESCRT-II complex are selected from one or more of Vps22, Vps25, Vps36, EPA30, EPA20 and EPA45;

[0015] The one or more proteins in the ESCRT-III complex are selected from one or more of Vps2, Vps20, Snf7, Vps24, Did2, Vps60, Ist1, CHMP2A, CHMP2B, CHMP6, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP5, CHMP1A, CHMP1B, CHMP7 and IST1;

[0016] The one or more proteins in the Vps4-Vta1 complex are selected from one or more of Vta1, Vps4, SKD1, SKD2 and LIP5;

[0017] The one or more proteins in the ALIX dimer are selected from one or more of Brol, ALIX and HD-PTP.

[0018] Optionally, the first subcellular structure is selected from one or more of lipid droplets, peroxisomes, lysosomes, mitochondria, centrosomes, Golgi bodies and microsomes.

[0019] Optionally, the fusion protein further comprises a fusion junction for connecting the localization peptide and the ESCRT protein; optionally, the amino acid sequence of the fusion junction is (GSG) n1 、(GGGGS) n2 、(G) n3 or (GS) n4 , where n1 to n4 are positive integers greater than or equal to 1.

[0020] Optionally, the first subcellular structure is a lipid droplet, and the localization peptide of the first subcellular structure is one or more of the following (A1) to (A3):

[0021] (A1) has the amino acid sequence shown in SEQ ID NO. 19;

[0022] (A2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO. 19;

[0023] The amino acid sequence (A3) is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO. 19, and has the function of localizing on the surface of the lipid droplet.

[0024] Optionally, the second protein is Snf7, and the Snf7 is one or more of the following (B1) to (B3):

[0025] (B1) having the amino acid sequence shown in SEQ ID NO. 20;

[0026] (B2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO. 20;

[0027] (B3) The amino acid sequence is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO. 20, and has the same function as the protein with the amino acid sequence shown in SEQ ID NO. 20.

[0028] Optionally, the fusion protein is one or more of the following (C1) to (C3):

[0029] (C1) has the amino acid sequence shown in SEQ ID NO. 21;

[0030] (C2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO. 21;

[0031] The (C3) amino acid sequence is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO.21, and has the same function as the protein with the amino acid sequence shown in SEQ ID NO.21.

[0032] Optionally, the second protein is STP22, and the STP22 is one or more of the following (D1) to (D3):

[0033] (D1) has the amino acid sequence shown in SEQ ID NO. 24;

[0034] (D2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO. 24;

[0035] The (D3) amino acid sequence is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO. 24, and has the same function as the protein with the amino acid sequence shown in SEQ ID NO. 24.

[0036] Optionally, the second protein is VPS25, and the VPS25 is one or more of the following (E1) to (E3):

[0037] (E1) has the amino acid sequence shown in SEQ ID NO. 26;

[0038] (E2) an amino acid sequence having no less than 80% homology to the amino acid sequence shown in SEQ ID NO. 26;

[0039] The (E3) amino acid sequence is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO. 26, and has the same function as the protein with the amino acid sequence shown in SEQ ID NO. 26.

[0040] Optionally, the second protein is VTA1, and the VTA1 is one or more of the following (F1) to (F3):

[0041] (F1) has the amino acid sequence shown in SEQ ID NO. 28;

[0042] (F2) an amino acid sequence having no less than 80% homology to the amino acid sequence shown in SEQ ID NO. 28;

[0043] The (F3) amino acid sequence is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO. 28, and has the same function as the protein with the amino acid sequence shown in SEQ ID NO. 28.

[0044] In a second aspect, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein, wherein the fusion protein comprises a localization peptide of a first subcellular structure and an ESCRT protein, wherein the ESCRT protein comprises one or more of the following (a) to (f):

[0045] (a) one or more proteins in the ESCRT-0 complex;

[0046] (b) one or more proteins in the ESCRT-I complex;

[0047] (c) one or more proteins in the ESCRT-II complex;

[0048] (d) one or more proteins in the ESCRT-III complex;

[0049] (e) one or more proteins in the Vps4-Vta1 complex;

[0050] (f) One or more proteins in the ALIX dimer.

[0051] Optionally, the nucleic acid molecule further comprises one or more of a promoter, an intron, an exon, a terminator and an enhancer.

[0052] Optionally, the nucleic acid molecule is one or more of the following (G1) to (G3):

[0053] (G1) has the nucleotide sequence shown in SEQ ID NO. 30;

[0054] (G2) The nucleotide sequence has no less than 80% homology with the nucleotide sequence shown in SEQ ID NO. 30;

[0055] The (G3) nucleotide sequence is obtained by substituting at least one nucleotide, and / or deleting at least one nucleotide, and / or adding at least one nucleotide from the nucleotide sequence shown in SEQ ID NO. 30, and has the function of encoding the fusion protein.

[0056] Optionally, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO.25, or the nucleotide sequence shown in SEQ ID NO.27, or the nucleotide sequence shown in SEQ ID NO.29.

[0057] In a third aspect, the present application provides a recombinant expression vector comprising a vector and a nucleic acid molecule loaded therein, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a fusion protein, wherein the fusion protein comprises a localization peptide of a first subcellular structure and an ESCRT protein, wherein the ESCRT protein comprises one or more of the following (a) to (f):

[0058] (a) one or more proteins in the ESCRT-0 complex;

[0059] (b) one or more proteins in the ESCRT-I complex;

[0060] (c) one or more proteins in the ESCRT-II complex;

[0061] (d) one or more proteins in the ESCRT-III complex;

[0062] (e) one or more proteins in the Vps4-Vta1 complex;

[0063] (f) One or more proteins in the ALIX dimer.

[0064] Optionally, the vector is selected from a plasmid, a phage, a phagemid, a cosmid, a virus, a yeast artificial chromosome or a bacterial artificial chromosome.

[0065] In a fourth aspect, the present application provides a recombinant comprising a host, and a nucleic acid molecule or a recombinant expression vector introduced into the host, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a fusion protein or the recombinant expression vector comprises a nucleotide sequence encoding a fusion protein, wherein the fusion protein comprises a localization peptide of a first subcellular structure and an ESCRT protein, wherein the ESCRT protein comprises one or more of the following (a) to (f):

[0066] (a) one or more proteins in the ESCRT-0 complex;

[0067] (b) one or more proteins in the ESCRT-I complex;

[0068] (c) one or more proteins in the ESCRT-II complex;

[0069] (d) one or more proteins in the ESCRT-III complex;

[0070] (e) one or more proteins in the Vps4-Vta1 complex;

[0071] (f) One or more proteins in the ALIX dimer.

[0072] Optionally, the host is yeast.

[0073] In a fifth aspect, the present application provides a method for preparing a fusion protein, wherein the recombinant described in any one of the fourth aspects is fermented and cultured to express and obtain the fusion protein.

[0074] Optionally, the method for introducing the nucleic acid molecule into the host comprises the following steps:

[0075] Providing the nucleic acid molecule, which comprises a first nucleotide sequence to a third nucleotide sequence connected in series, wherein the first nucleotide sequence is an upstream homology arm sequence of the integration site in the host genome, the second nucleotide sequence is a nucleotide sequence encoding the fusion protein, and the third nucleotide sequence is a downstream homology arm sequence of the integration site in the host genome; and

[0076] The nucleic acid molecule is transformed into the host, and homologous recombination occurs between the nucleic acid molecule and the genome of the host.

[0077] In a sixth aspect, the present application provides a method for preparing a metabolite, comprising the steps of: fermenting and culturing the recombinant described in any one of the fourth aspects to express and produce the fusion protein, thereby secreting the second subcellular structure storing the target metabolite to the extracellular space through the ESCRT pathway; wherein the first subcellular structure is the same as the second subcellular structure. Beneficial effects

[0078] In the fusion protein of the present application, the fusion protein comprises a localization peptide and an ESCRT protein of a first subcellular structure. The fusion protein can be used to prepare metabolites. For a host capable of expressing the fusion protein, the fusion protein can be localized on the surface of a second subcellular structure identical to the first subcellular structure in the host body, thereby allowing the second subcellular structure storing the target metabolite to enter the sorting link through the ESCRT pathway, and eventually enter the exosomes (vesicles that exist endogenously in the cell and secrete proteins and biological substances to the outside), and be secreted to the extracellular space through the exosomes, thereby effectively increasing the extracellular yield of the target metabolite, and omitting the cell lysis process, which is beneficial to reducing the synthesis cost of the target metabolite and improving the purity of the target metabolite. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0080] FIG1 is a schematic diagram of the composition of the fusion protein provided in the examples of the present application;

[0081] FIG2 is a schematic diagram of the composition of the target gene fragment provided in the examples of this application;

[0082] FIG3 is a graph showing the extracellular yield of carotenoids produced by various recombinant and control fermentations in the experimental examples of this application;

[0083] FIG4 is a comparison picture of the supernatant of the Sc-1 recombinant and the control in the experimental example of this application.

[0084] Implementation Methods of the Application

[0085] The terms used in this application are defined as follows:

[0086] As used in this application, the Chinese name of "ESCRT" is endosomal sorting complex required for transport, and its full English name is endosomal sorting complex required for transport, which refers to the molecular machine that completes the invagination of the endosome membrane to form a multi-vesicular body (MVB) in eukaryotic cells. ESCRT is widely present in various tissue cells of the human body, such as muscle tissue, neural tissue, epithelial tissue, etc., and participates in important life processes such as cytokinesis, virus budding, apoptosis, autophagy, protein quality control, PMR, and nuclear membrane remodeling. ESCRT contains 6 complexes, namely ESCRT-0 complex, ESCRT-Ⅰ complex, ESCRT-Ⅱ complex, ESCRT-III complex, Vps4-Vta1 complex and ALIX dimer. The ESCRT systems of humans and yeast are highly homologous, and the domains or motifs that play their specific roles are similar.

[0087] As used in this application, "plurality" refers to two or more, for example, two, three, four, five, six, seven, eight, nine, ten, etc.

[0088] As used in this application, "and / or" means that the selection range includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, and the said any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, a combination of any two or any three of A, B, C, and D, and also includes a four-item combination of A, B, C, and D (that is, a technical solution connected by "logical AND").

[0089] As used in this application, "homology" refers to the "identity" between two nucleic acid or amino acid sequences, and its percentage represents the statistically significant percentage of identical nucleotides or amino acid residues between the two sequences to be compared obtained after the best alignment (best alignment), and the differences between the two sequences are randomly distributed over their entire length. The percentage of identity or consistency means the percentage of identical nucleotides or amino acid residues between the two sequences to be compared obtained after the best alignment (best alignment), which is purely statistical, and the differences between the two sequences are randomly distributed and distributed over their entire length. Usually, the best alignment (best alignment) is the alignment with the highest percentage of identity between the two sequences to be compared, which can be performed manually or using a sequence alignment tool (such as Blast or other online sequence alignment software).

[0090] In the present application embodiment, at least 80% homology can be understood as at least greater than 81%, or at least greater than 82%, or at least greater than 83%, or at least greater than 84%, or at least greater than 84%, or at least greater than 85%, or at least greater than 86%, or at least greater than 87%, or at least greater than 88%, or at least greater than 89%, or at least greater than 90%, or at least greater than 91%, or at least greater than 92%, or at least greater than 93%, or at least greater than 94%, or at least greater than 95%, or at least greater than 96%, or at least greater than 97%, or at least greater than 98%, or at least greater than 99% sequence homology, examples of which are 80%, 81%, 82%, 83%, 84%, or at least greater than 96%. %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, the corresponding homology value is an integer; it can also be further understood as 80.1%, 81.2%, 82.3%, 83.4%, 84.5%, 85.6%, 86.7%, 87.8%, 88.9%, 89.8%, 90.3%, 91.7%, 92.2%, 93.5%, 94.8%, 95.9%, 96.6%, 97.5%, 98.4% or 99.9%, but the sequence identity is less than 100%, the corresponding homology value is a decimal.

[0091] As used in this application, "nucleic acid molecule" refers to a biomacromolecule compound comprising a nucleotide sequence encoding a fusion protein in this application. The nucleic acid molecule of the embodiment of the present application can be, for example, a deoxyribonucleic acid (DNA) fragment synthesized by polymerase chain reaction (PCR), or a fragment produced by any one or more of ligation, cleavage, endonuclease action, and exonuclease action, and can be single-stranded or double-stranded. In the embodiment of the present application, the nucleic acid molecule includes a coding region and a non-coding region, and the non-coding region optionally includes a promoter, introns, exons, terminators, enhancers or other expression regulatory elements, and the nucleic acid molecule can also include an enzyme cleavage site. In some embodiments of the present application, the promoter can be a promoter in any Saccharomyces cerevisiae, preferably ADH2; and / or, the terminator can be a terminator in any Saccharomyces cerevisiae, such as the CYC1 terminator.

[0092] As used in this application, "recombinant expression vector" refers to a nucleic acid molecule construct that contains an exogenous nucleic acid fragment operably linked to an appropriate control sequence that can achieve expression of the exogenous nucleic acid fragment in a suitable host. Recombinant expression vectors include, for example: (1) a collection of genetic elements that regulate the expression of exogenous nucleic acid fragments, such as promoters, enhancers, enhancers, silencers, etc.; (2) a structure or coding sequence that is transcribed into mRNA and translated into protein. Those skilled in the art can use widely known methods to construct recombinant expression vectors, including but not limited to double enzyme digestion construction methods or homologous recombination construction methods. The expression vector may contain one or more selectable marker genes.

[0093] As used in this application, "vector" refers to a tool that uses genetic engineering and recombinant technology to transfer a target nucleic acid molecule into a host for amplification and expression. The vector can be, for example, a plasmid, a phage, a phagemid, a cosmid, a virus, a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), an Agrobacterium pTi plasmid, etc. It should be noted that those skilled in the art may use appropriate vectors to implement the technical solutions of this application, but such substitutions do not deviate from the scope of this application, and this application shall include such alternative vectors.

[0094] As used in this application, "host" refers to a type of recipient microorganism that can accept exogenous nucleic acid (e.g., the nucleic acid molecule of this application) by a specific means, including but not limited to at least one of conjugation, electroporation, chemical transformation, transduction, transfection or ultrasonic transformation.

[0095] This application studies a fusion protein and its application in preparing metabolites. As shown in FIG1 , the fusion protein comprises a localization peptide of a first subcellular structure and an ESCRT protein, wherein the ESCRT protein comprises one or more of the following (a) to (f):

[0096] (a) one or more proteins in the ESCRT-0 complex;

[0097] (b) one or more proteins in the ESCRT-I complex;

[0098] (c) one or more proteins in the ESCRT-II complex;

[0099] (d) one or more proteins in the ESCRT-III complex;

[0100] (e) one or more proteins in the Vps4-Vta1 complex;

[0101] (f) One or more proteins in the ALIX dimer.

[0102] The structural composition of each complex in the ESCRT system is shown in Table 1 below:

[0103] Table 1 Structural composition of each complex in the ESCRT system

[0104] Note: CHMP2A and CHMP2B correspond to two Vps2 proteins, CHMP4A, CHMP4B and CHMP4C correspond to three Snf7 proteins, and CHMP1A and CHMP1B correspond to two Did2 proteins.

[0105] It can be understood that the ESCRT protein in the fusion protein can be a protein, and the protein can be a protein in the ESCRT-0 complex, a protein in the ESCRT-I complex, a protein in the ESCRT-II complex, a protein in the ESCRT-III complex, a protein in the Vps4-Vta1 complex or a protein in the ALIX dimer, and a protein in each complex can correspond to any one protein selected from the corresponding complex in Table 1, for example: the ESCRT protein in the fusion protein can be a protein in the ESCRT-III complex, which can be selected from Vps2, Vps20, Snf7, Vps24, Did2, Vps60, Ist1, CHMP2A, CHMP2B, CHMP6, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP5, CHMP1A, CHMP1B, CHMP7 or IST1.

[0106] The ESCRT protein in the fusion protein can also be two or more proteins, and the two or more proteins can all be proteins of the same complex, or proteins of different complexes, or proteins of the same complex. The two or more proteins can be connected in series through a linker. As an example, the ESCRT protein in the fusion protein comprises two proteins, both of which can be proteins in the ESCRT-III complex, or the two proteins can be one protein in the ESCRT-I complex and one protein in the ESCRT-II complex. As an example, the ESCRT protein in the fusion protein comprises three proteins, all of which can be proteins in the ESCRT-III complex, or the three proteins can be one protein in the ESCRT-I complex, one protein in the ESCRT-II complex, and one protein in the ESCRT-III complex.

[0107] In some embodiments of the present application, the one or more proteins in the ESCRT-0 complex are selected from one or more of Vps27, Hse1, HRS, STAM1 and STAM2; and / or, the one or more proteins in the ESCRT-I complex are selected from one or more of Vps23, Vps28, Vps37, Mvb12, STP22, TSG101, VPS28, VPS37A, VPS37B, VPS37C, VPS37D, MVB12A, MVB12B, UBAP1, UBA1L and UMAD1; and / or, the one or more proteins in the ESCRT-II complex are selected from one or more of Vps22, Vps25, Vps36, EPA30, EPA20 and EPA45. one or more of; and / or, one or more proteins in the ESCRT-III complex are selected from one or more of Vps2, Vps20, Snf7, Vps24, Did2, Vps60, Ist1, CHMP2A, CHMP2B, CHMP6, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP5, CHMP1A, CHMP1B, CHMP7 and IST1; and / or, one or more proteins in the Vps4-Vta1 complex are selected from one or more of Vta1, Vps4, SKD1, SKD2 and LIP5; and / or, one or more proteins in the ALIX dimer are selected from one or more of Brol, ALIX and HD-PTP.

[0108] In some embodiments of the present application, referring to FIG1 , the fusion protein further comprises a fusion linker for connecting the localization peptide of the first subcellular structure with the ESCRT protein, which can be a fusion linker commonly used in the art. The amino acid sequence of the fusion linker includes but is not limited to (GSG)n1 、(GGGGS) n2 、(G) n3 or (GS) n4 , wherein n1 to n4 are positive integers greater than or equal to 1. The localization peptide and the ESCRT protein connected by the fusion junction still have independent functions, and the length of the fusion junction is, for example, 3 to 15 amino acids.

[0109] It should be noted that in the fusion protein, the order in which the localization peptide of the first subcellular structure and the ESCRT protein are linked is not specifically limited, and the localization peptide of the first subcellular structure can be closer to the N-terminus or C-terminus. When the ESCRT protein comprises multiple proteins, the order in which the multiple proteins are linked is also not specifically limited.

[0110] In some embodiments of the present application, the first subcellular structure is selected from one or more of lipid droplets, peroxisomes, lysosomes, mitochondria, centrosomes, Golgi bodies, and microsomes.

[0111] In some embodiments of the present application, the first subcellular structure is a lipid droplet, and the localization peptide of the first subcellular structure is one or more of the following (A1) to (A3):

[0112] (A1) has the amino acid sequence shown in SEQ ID NO. 19;

[0113] (A2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO. 19;

[0114] The amino acid sequence (A3) is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO. 19, and has the function of localizing on the surface of the lipid droplet.

[0115] Regarding (A3), it should be noted that the localization peptide of the first subcellular structure can be a variant form of the amino acid sequence shown in SEQ ID NO. 19, which has the function of localizing on the surface of the lipid droplet and has a small difference from the amino acid sequence shown in SEQ ID NO. 19. These variant forms include, but are not limited to: one or more (usually 1 to 50, such as 1 to 30, such as 1 to 20, such as 1 to 10, such as 1 to 8, and such as 1 to 6) amino acid deletions, insertions, and / or substitutions, and the addition of one or more (usually within 20, such as within 10, and such as within 6) amino acids at the C-terminus and / or N-terminus. Those skilled in the art know that when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed; in addition, the addition of one or more amino acids at the C-terminus and / or N-terminus generally does not change the function of the protein.

[0116] In some embodiments of the present application, the second protein is Snf7, and Snf7 is one or more of the following (B1) to (B3):

[0117] (B1) having the amino acid sequence shown in SEQ ID NO. 20;

[0118] (B2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO. 20;

[0119] (B3) The amino acid sequence is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO. 20, and has the same function as the protein with the amino acid sequence shown in SEQ ID NO. 20.

[0120] Regarding (B3), it should be noted that Snf7 may be a variant form of the amino acid sequence shown in SEQ ID NO.20, which has the same or similar function as Snf7 and has a small difference from the amino acid sequence shown in SEQ ID NO.20. These variant forms include but are not limited to: deletion, insertion and / or substitution of one or more (usually 1 to 50, for example 1 to 30, for example 1 to 20, for example 1 to 10, for example 1 to 8, for example 1 to 6) amino acids, and addition of one or more (usually within 20, for example within 10, for example within 6) amino acids at the C-terminus and / or N-terminus.

[0121] Furthermore, the fusion protein is one or more of the following (C1) to (C3):

[0122] (C1) has the amino acid sequence shown in SEQ ID NO. 21;

[0123] (C2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO. 21;

[0124] The (C3) amino acid sequence is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO.21, and has the same function as the protein with the amino acid sequence shown in SEQ ID NO.21.

[0125] Regarding (C3), it should be noted that the fusion protein may be a variant form of the amino acid sequence shown in SEQ ID NO. 21, and may have a small difference from the amino acid sequence shown in SEQ ID NO. 21, and the fusion protein has the function of promoting the secretion of the substance stored in the first subcellular structure to the extracellular space. The variant forms include, but are not limited to: deletion, insertion and / or substitution of one or more (usually 1 to 50, for example, 1 to 30, for example, 1 to 20, for example, 1 to 10, for example, 1 to 8, for example, 1 to 6) amino acids, and addition of one or more (usually within 20, for example, within 10, for example, within 6) amino acids to the C-terminus and / or N-terminus.

[0126] As an alternative embodiment, in some embodiments of the present application, the second protein is STP22, and STP22 is one or more of the following (D1) to (D3):

[0127] (D1) has the amino acid sequence shown in SEQ ID NO. 24;

[0128] (D2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO. 24;

[0129] The (D3) amino acid sequence is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO. 24, and has the same function as the protein with the amino acid sequence shown in SEQ ID NO. 24.

[0130] Regarding (D3), it should be noted that STP22 may be a variant form of the amino acid sequence shown in SEQ ID NO. 24, and the variant form has the same or similar function as STP22 and has a small difference from the amino acid sequence shown in SEQ ID NO. 24. These variant forms include but are not limited to: deletion, insertion and / or substitution of one or more (usually 1 to 50, for example 1 to 30, for example 1 to 20, for example 1 to 10, for example 1 to 8, for example 1 to 6) amino acids, and addition of one or more (usually within 20, for example within 10, for example within 6) amino acids at the C-terminus and / or N-terminus.

[0131] As an alternative embodiment, in some embodiments of the present application, the second protein is VPS25, and VPS25 is one or more of the following (E1) to (E3):

[0132] (E1) has the amino acid sequence shown in SEQ ID NO. 26;

[0133] (E2) an amino acid sequence having no less than 80% homology to the amino acid sequence shown in SEQ ID NO. 26;

[0134] The (E3) amino acid sequence is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO. 26, and has the same function as the protein with the amino acid sequence shown in SEQ ID NO. 26.

[0135] Regarding (E3), it should be noted that VPS25 may be a variant form of the amino acid sequence shown in SEQ ID NO. 26, which has the same or similar function as VPS25 and has a small difference from the amino acid sequence shown in SEQ ID NO. 26. These variant forms include but are not limited to: one or more (usually 1 to 50, for example 1 to 30, for example 1 to 20, for example 1 to 10, for example 1 to 8, for example 1 to 6) amino acid deletions, insertions and / or substitutions, and addition of one or more (usually within 20, for example within 10, for example within 6) amino acids at the C-terminus and / or N-terminus.

[0136] As an alternative embodiment, in some embodiments of the present application, the second protein is VTA1, and VTA1 is one or more of the following (F1) to (F3):

[0137] (F1) has the amino acid sequence shown in SEQ ID NO. 28;

[0138] (F2) an amino acid sequence having no less than 80% homology to the amino acid sequence shown in SEQ ID NO. 28;

[0139] The (F3) amino acid sequence is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid to the amino acid sequence shown in SEQ ID NO. 28, and has the same function as the protein with the amino acid sequence shown in SEQ ID NO. 28.

[0140] Regarding (F3), it should be noted that VTA1 can be a variant form of the amino acid sequence shown in SEQ ID NO.28, which has the same or similar function as VTA1 and has a small difference from the amino acid sequence shown in SEQ ID NO.28. These variant forms include but are not limited to: one or more (usually 1 to 50, for example 1 to 30, for example 1 to 20, for example 1 to 10, for example 1 to 8, for example 1 to 6) amino acid deletions, insertions and / or substitutions, and addition of one or more (usually within 20, for example within 10, for example within 6) amino acids at the C-terminus and / or N-terminus.

[0141] In the fusion protein of the embodiment of the present application, the fusion protein comprises a localization peptide and an ESCRT protein of a first subcellular structure. For a host capable of expressing the fusion protein, the fusion protein can be localized on the surface of a second subcellular structure identical to the first subcellular structure in the host body, thereby allowing the second subcellular structure storing the target metabolite to enter the sorting link through the ESCRT pathway, and eventually enter the exosomes (vesicles that exist endogenously in the cell and secrete proteins and biological substances to the outside), and be secreted to the extracellular space through the exosomes, thereby effectively increasing the extracellular yield of the target metabolite, and omitting the cell lysis process, which is beneficial to reducing the synthesis cost of the target metabolite and improving the purity of the target metabolite.

[0142] The embodiments of the present application also provide a nucleic acid molecule comprising a nucleotide sequence encoding any one of the fusion proteins described above.

[0143] In some embodiments of the present application, the nucleic acid molecule is one or more of the following (G1) to (G3):

[0144] (G1) has the nucleotide sequence shown in SEQ ID NO. 30;

[0145] (G2) The nucleotide sequence has no less than 80% homology with the nucleotide sequence shown in SEQ ID NO. 30;

[0146] The (G3) nucleotide sequence is obtained by substituting at least one nucleotide, and / or deleting at least one nucleotide, and / or adding at least one nucleotide from the nucleotide sequence shown in SEQ ID NO. 30, and has the function of encoding the fusion protein.

[0147] Regarding (G3), it should be noted that the nucleic acid molecule may be a variant of a polynucleotide having a nucleotide sequence as shown in SEQ ID NO. 30, and has the function of encoding one or more fusion proteins of (C1) to (C3). The variant may be a naturally occurring allelic variant or a non-naturally occurring variant, including but not limited to substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may have one or more nucleotide substitutions, deletions, or insertions that do not substantially alter the function of the encoded protein.

[0148] In some embodiments of the present application, the nucleic acid molecule is one or more of the following (H1) to (H3):

[0149] (H1) has the nucleotide sequence shown in SEQ ID NO. 25;

[0150] (H2) the nucleotide sequence has no less than 80% homology with the nucleotide sequence shown in SEQ ID NO. 25;

[0151] The (H3) nucleotide sequence is obtained by substituting at least one nucleotide, and / or deleting at least one nucleotide, and / or adding at least one nucleotide from the nucleotide sequence shown in SEQ ID NO. 25, and has the function of encoding the fusion protein.

[0152] Regarding (H3), it should be noted that the nucleic acid molecule may be a variant of a polynucleotide having the nucleotide sequence shown in SEQ ID NO. 25, and has the function of encoding the fusion protein PLIN1-STP22. The variant may be a naturally occurring allelic variant or a non-naturally occurring variant, including but not limited to substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may have one or more nucleotide substitutions, deletions, or insertions that do not substantially alter the function of the encoded protein.

[0153] In some embodiments of the present application, the nucleic acid molecule is one or more of the following (I1) to (I3):

[0154] (I1) having the nucleotide sequence shown in SEQ ID NO. 27;

[0155] (I2) the nucleotide sequence has no less than 80% homology with the nucleotide sequence shown in SEQ ID NO. 27;

[0156] (I3) The nucleotide sequence is obtained by replacing at least one nucleotide, and / or deleting at least one nucleotide, and / or adding at least one nucleotide from the nucleotide sequence shown in SEQ ID NO. 27, and has the function of encoding the fusion protein.

[0157] Regarding (I3), it should be noted that the nucleic acid molecule may be a variant of a polynucleotide having a nucleotide sequence as shown in SEQ ID NO. 27, and having the function of encoding the fusion protein PLIN1-VPS25. The variant may be a naturally occurring allelic variant or a non-naturally occurring variant, including but not limited to substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the protein it encodes.

[0158] In some embodiments of the present application, the nucleic acid molecule is one or more of the following (J1) to (J3):

[0159] (J1) having the nucleotide sequence shown in SEQ ID NO. 29;

[0160] (J2) the nucleotide sequence has no less than 80% homology with the nucleotide sequence shown in SEQ ID NO. 29;

[0161] (J3) The nucleotide sequence is obtained by substituting at least one nucleotide, and / or deleting at least one nucleotide, and / or adding at least one nucleotide from the nucleotide sequence shown in SEQ ID NO. 29, and has the function of encoding the fusion protein.

[0162] Regarding (J3), it should be noted that the nucleic acid molecule may be a variant of a polynucleotide having the nucleotide sequence set forth in SEQ ID NO. 29, and may function to encode the fusion protein PLIN1-VTA1. The variant may be a naturally occurring allelic variant or a non-naturally occurring variant, including but not limited to substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may contain one or more nucleotide substitutions, deletions, or insertions that do not substantially alter the function of the encoded protein.

[0163] It should be noted that, for example, a nucleic acid molecule includes a nucleotide sequence X, a nucleotide sequence Y, and a nucleotide sequence Z in series, wherein the nucleotide sequence X is used to encode a localization peptide for a first subcellular structure, the nucleotide sequence Y is used to encode a fusion junction, and the nucleotide sequence Z is used to encode an ESCRT protein. The expression "in series" does not limit the order of the nucleotide sequence X, the nucleotide sequence Y, and the nucleotide sequence Z. For example, the nucleotide sequence X may be closer to the 5' end than the nucleotide sequence Z, and the nucleotide sequence X may also be closer to the 3' end than the nucleotide sequence Z. It should be noted that the order of the series connection only brings about different operations and does not affect the expression effect.

[0164] An embodiment of the present application further provides a recombinant expression vector, comprising a vector and any one of the aforementioned nucleic acid molecules, wherein the nucleic acid molecule is loaded in the vector.

[0165] In the recombinant expression vector, the nucleic acid molecule can be a single copy or multiple copies, for example, the nucleic acid molecule can be a double copy. When the nucleic acid molecule is multiple copies, they can be sequentially linked end to end to form a tandem repeat sequence.

[0166] The embodiments of the present application also provide a recombinant, which comprises a host, and a nucleic acid molecule as described above, or a recombinant expression vector as described above, introduced into the host.

[0167] In some embodiments of the present application, the host is a eukaryotic organism, such as yeast, including but not limited to Saccharomyces cerevisiae, Saccharomyces pastoris, Saccharomyces boulardii, Saccharomyces fermentans, Saccharomyces paradoxus, Saccharomyces vitis, Saccharomyces bayanus, Schizosaccharomyces, Kluyveromyces, Torulospora, Pichia, Zygosaccharomyces, Brettanomyces, or Metschneiderian. Among them, the genus Schizosaccharomyces includes but is not limited to Schizosaccharomyces pombe, Schizosaccharomyces japonicus, Schizosaccharomyces octospora, or Schizosaccharomyces psychrophilus; the genus Torulospora includes but is not limited to Saccharomyces deboer; the genus Kluyveromyces includes but is not limited to Kluyveromyces marxianus; the genus Pichia includes but is not limited to Pichia stipitis, Pichia pastoris, or Pichia angus; the genus Zygosaccharomyces includes but is not limited to Zygosaccharomyces bailii; the genus Brettanomyces includes but is not limited to Brettanomyces intermedius, Brettanomyces bruxellensis, Brettanomyces heterotypicus, Brettanomyces bantuensis, Brettanomyces natterii, Brettanomyces nanus, Dekkera bruxellensis, or Dekkera heterotypicus; the genus Metschneiderian includes but is not limited to Issaffini or Klerkers. As an example, the host is Saccharomyces cerevisiae.

[0168] It should be noted that the host can be a wild type microorganism or a microorganism obtained by natural mutation or artificial modification of the wild type. In the present embodiment, the host is Saccharomyces cerevisiae YB / I / E extra I, which is a recombinant strain that has been artificially modified to produce high β-carotene.

[0169] This application also provides a method for preparing a fusion protein, comprising the steps of fermenting any of the recombinant proteins described above to express the fusion protein. Depending on the selected host, the culture medium used in the fermentation culture can be selected from various conventional culture media, and the culture is carried out under conditions suitable for the growth of the host. For example, using Saccharomyces cerevisiae as the host, the culture medium can be YPD, etc., and the culture temperature can be 30°C, with aerobic culture.

[0170] The fusion protein can be expressed in the host cell or on the cell membrane or secreted into the extracellular space. If necessary, the fusion protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art and include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations thereof.

[0171] In some embodiments of the present application, the method of introducing any of the aforementioned nucleic acid molecules into a host comprises the following steps:

[0172] S1. Providing a nucleic acid molecule, the nucleic acid molecule comprising a first nucleotide sequence to a third nucleotide sequence connected in series, wherein the first nucleotide sequence is an upstream homology arm sequence of the integration site in the host genome, the second nucleotide sequence is a nucleotide sequence encoding a fusion protein, and the third nucleotide sequence is a downstream homology arm sequence of the integration site in the host genome;

[0173] S2. The nucleic acid molecule of step S1 is transformed into a host, and homologous recombination occurs between the nucleic acid molecule and the host genome.

[0174] In step S1, when the host is Saccharomyces cerevisiae, the integration site may be, for example, 511b.

[0175] In step S2, "transformation" can be performed using conventional techniques well known to those skilled in the art, and an appropriate transformation method is selected based on the selected host. For example, when the host is Saccharomyces cerevisiae, lithium acetate transformation can be selected. To facilitate screening of positive clones, the nucleic acid molecule can be transformed into the host together with the CRISPR-Cas system. The CRISPR-Cas system can be used as a screening system, thereby improving the problem of difficult screening in recombineering engineering.

[0176] The present application also provides a method for preparing a metabolite, comprising the steps of: fermenting and culturing any one of the recombinants described above to express and produce a fusion protein, and secreting the second subcellular structure storing the target metabolite to the extracellular space through the ESCRT pathway, wherein the first subcellular structure is the same as the second subcellular structure.

[0177] It should be noted that microorganisms are often used for efficient heterologous synthesis of natural products (primarily microbial metabolites). However, the synthesized natural products are stored intracellularly (e.g., within subcellular structures) and are not secreted extracellularly or in very small amounts, often requiring cell disruption to obtain. Taking lipid droplets as an example, lipid droplets are primarily composed of neutral fats and a small amount of phospholipids, triglycerides, proteins, and other bioactive substances. They play a role in energy metabolism, lipid metabolism, cell membrane synthesis and transport, material storage, and the delivery of bioactive molecules. Lipid droplets are a storage site for fat-soluble compounds, including but not limited to carotenoids, withanolides, ubiquinones, polyterpenoids, sterols, and biofuels (e.g., squalene).

[0178] In the preparation method of the metabolite of the present application, the recombinant expresses a fusion protein comprising a localization peptide of a first subcellular structure and an ESCRT protein, and the first subcellular structure is identical to the second subcellular structure, so that the fusion protein can be localized on the surface of the second subcellular structure, thereby allowing the second subcellular structure storing the target metabolite to enter the sorting link through the ESCRT pathway, and eventually enter the exosomes (vesicles that exist endogenously in the cell and secrete proteins and biological substances to the outside), and be secreted to the extracellular space through the exosomes, thereby effectively increasing the extracellular yield of the target metabolite, and omitting the cell lysis process, which is beneficial to reducing the synthesis cost of the target metabolite and improving the purity of the target metabolite.

[0179] It is understandable that in order to further increase the extracellular production of the target metabolite, a microorganism with high production of the target metabolite can be selected as the host of the recombinant.

[0180] The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of this application. The experimental methods in the following examples where specific conditions are not specified are generally based on conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0181] Unless otherwise defined or indicated, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art, and the materials or reagents used in the examples and comparative examples of the present application are commercially available. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for exemplary purposes only and do not limit the content of this application. The terms "first," "second," "third," or similar expressions are used for descriptive purposes only and are not to be construed as indicating or implying relative importance, nor as indicating or implying a sequential order.

[0182] I. Description of reagents and materials involved in the examples of this application:

[0183] The primers and gene fragments involved in the examples of this application were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0184] The PCR reaction reagents involved in the examples of this application were purchased from Nanjing Novozymes Biotechnology Co., Ltd., among which the model of 2×Phanta Max Master Mix was P515;

[0185] The Saccharomyces cerevisiae involved in the examples of the present application is YB / I / E extra I, which is derived from the document Verwaal, R., Wang, J., Meijnen, JP, Visser, H., Sandmann, G., van den Berg, JA, & van Ooyen, AJ (2007). High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous. Appl Environ Microbiol, 73(13), 4342-4350. https: / / doi.org / 10.1128 / AEM.02759-06.

[0186] The construction method of the pCUT (511b-ura) plasmid involved in the examples of the present application is derived from the document Reider Apel, A., d'Espaux, L., Wehrs, M., Sachs, D., Li, RA, Tong, GJ, Garber, M., Nnadi, O., Zhuang, W., Hillson, NJ, Keasling, JD, & Mukhopadhyay, A. (2017). A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. Nucleic Acids Res, 45(1), 496-508. (https: / / doi.org / 10.1093 / nar / gkw1023).

[0187] The single-stranded fish sperm DNA involved in the examples of this application was purchased from Beijing Solebow Technology Co., Ltd., model number H1060.

[0188] The YPD medium involved in the examples of the present application was prepared by ourselves, and the formula of the YPD liquid medium was: 10 g / L yeast extract, 20 g / L peptone and 2 g / L glucose.

[0189] The SC-URA culture medium involved in the examples of this application was purchased from Beijing Coolabo Technology Co., Ltd., model number PM2273.

[0190] II. Specific information of the primers involved in the examples of this application is shown in Table 2 below:

[0191] Table 2 Primer list

[0192] III. Detection Method of β-Carotene in the Supernatant of the Experimental Examples of the Present Application

[0193] The present invention uses high performance liquid chromatography (HPLC) to detect the content of β-carotene in the supernatant. The external standard method is used to quantify the β-carotene content by peak area. β-carotene standard (purchased from Sigma) is used as the qualitative and quantitative standard for β-carotene in the supernatant. The HPLC operating conditions are as follows:

[0194] (1) Preparation of injection solution: Filter the supernatant with a 0.22 μm filter. The filtered supernatant is the injection solution, and 5 μL is injected each time.

[0195] (2) Chromatographic column: YMC Carotenoid column, 4.6 mm × 250 mm, 5 μm;

[0196] (3) Preparation of mobile phase: Anhydrous acetonitrile and water were mixed in a volume ratio of 90:10 to prepare phase A; phase B was methyl tert-butyl ether (MTBE).

[0197] (4) Flow rate: 1 mL / min;

[0198] (6) Column temperature: 30°C;

[0199] (7) UV detection wavelength: 450nm.

[0200] The technical solutions and beneficial effects of the present invention are further illustrated below with reference to embodiments, comparative examples and experimental examples.

[0201] Example 1

[0202] This example provides a recombinant Sc-1 and a method for constructing the same. The recombinant Sc-1 is capable of expressing a fusion protein PLIN1-Snf7, wherein the fusion protein PLIN1-Snf7 has the amino acid sequence shown in SEQ ID NO.21. The fusion protein PLIN1-Snf7 comprises a lipid droplet localization peptide and a Snf7 protein in the ESCRT-III complex, wherein the lipid droplet localization peptide has the amino acid sequence shown in SEQ ID NO.19, and the Snf7 protein has the amino acid sequence shown in SEQ ID NO.20.

[0203] 1.1. Synthesis of target gene fragment encoding fusion protein PLIN1-Snf7

[0204] S1.11. Synthesis of a first gene fragment: Using human cDNA (NCBI accession number: NC_000015.10) as a template, PCR amplification was performed on the template using primer pair F1-R1, wherein the nucleotide sequence of F1 is shown in SEQ ID NO. 1, and the nucleotide sequence of R1 is shown in SEQ ID NO. 2. The PCR amplification product was purified and recovered by agarose gel electrophoresis to obtain a first gene fragment. The first gene fragment comprised a tandem DH2 promoter homology sequence and a nucleotide sequence encoding a lipid droplet localization peptide. The nucleotide sequence of the first gene fragment is shown in SEQ ID NO. 3.

[0205] S1.12. Synthesis of a second gene fragment: Using the Saccharomyces cerevisiae genome as a template, perform PCR amplification on the template using primer pair F2-R2, wherein the nucleotide sequence of F2 is shown in SEQ ID NO. 4, and the nucleotide sequence of R2 is shown in SEQ ID NO. 5. Purify and recover the PCR amplification product by agarose gel electrophoresis to obtain a second gene fragment. The second gene fragment comprises a nucleotide sequence encoding a lipid droplet localization peptide, a nucleotide sequence encoding a fusion junction, a nucleotide sequence encoding the Snf7 protein, and a CYC1 terminator homologous sequence, which are sequentially linked in series. The nucleotide sequence of the second gene fragment is shown in SEQ ID NO. 6.

[0206] S1.13. Synthesis of the third gene fragment: Using the Saccharomyces cerevisiae genome as a template, PCR amplification was performed on the template using primer pair F3-R3, wherein the nucleotide sequence of F3 is shown in SEQ ID NO. 7, and the nucleotide sequence of R3 is shown in SEQ ID NO. 8. The PCR amplification product was purified and recovered by agarose gel electrophoresis to obtain the third gene fragment. The third gene fragment comprises a tandem homologous sequence upstream of integration site 511b and the ADH2 promoter. The nucleotide sequence of the third gene fragment is shown in SEQ ID NO. 9.

[0207] S1.14. Synthesis of the fourth gene fragment: Using the Saccharomyces cerevisiae genome as a template, PCR amplification was performed on the template using primer pair F4-R4, wherein the nucleotide sequence of F4 is shown in SEQ ID NO. 10, and the nucleotide sequence of R4 is shown in SEQ ID NO. 11. The PCR amplification product was purified and recovered by agarose gel electrophoresis to obtain a fourth gene fragment. The fourth gene fragment comprises a tandem CYC1 terminator and a homologous sequence downstream of integration site 511b. The nucleotide sequence of the fourth gene fragment is shown in SEQ ID NO. 12.

[0208] S1.15. Synthesis of the fifth gene fragment: Using the Saccharomyces cerevisiae genome as a template, PCR amplification was performed on the template using primer pair F5-R5, wherein the nucleotide sequence of F5 is shown in SEQ ID NO. 13, and the nucleotide sequence of R5 is shown in SEQ ID NO. 14. The PCR amplification product was purified and recovered by agarose gel electrophoresis to obtain the fifth gene fragment. The fifth gene fragment includes the upstream homology arm sequence of integration site 511b, and the nucleotide sequence of the fifth gene fragment is shown in SEQ ID NO. 15.

[0209] S1.16. Synthesis of the sixth gene fragment: Using the Saccharomyces cerevisiae genome as a template, PCR amplification was performed on the template using primer pair F6-R6, wherein the nucleotide sequence of F6 is shown in SEQ ID NO. 16, and the nucleotide sequence of R6 is shown in SEQ ID NO. 17. The PCR amplification product was purified and recovered by agarose gel electrophoresis to obtain the sixth gene fragment. The sixth gene fragment includes the homology arm sequence downstream of integration site 511b, and the nucleotide sequence of the sixth gene fragment is shown in SEQ ID NO. 18.

[0210] S1.17, using primers F5 and R6 as primer pairs, the first to sixth gene segments were subjected to fusion PCR to obtain the target gene segment, as shown in FIG2 , the target gene segment comprises the upstream homology arm sequence (511b-HoL) of the integration site 511b, the ADH2 promoter (P ADH2 ), a nucleotide sequence encoding a lipid droplet localization peptide (PLIN1 (1-192)), a nucleotide sequence encoding a fusion junction (linker), a nucleotide sequence encoding Snf7 protein (Sc_SNF7), a CYC1 terminator (T CYC1 ) and the upstream homology arm sequence of the integration site 511b (511b-HoR).

[0211] The target gene fragment has a nucleotide sequence as shown in SEQ ID NO. 30; in steps S1.11 to S1.16, the PCR reaction system is shown in Table 3 below:

[0212] Table 3 PCR reaction system list

[0213] Compared with the PCR reaction system in Table 1, the difference between the reaction system of the fusion PCR in step S1.17 is that the total amount of the first to sixth gene fragments is 1.2 μL, the amount of each gene fragment is 0.2 μL, and the amount of double-distilled water is 19.8 μL.

[0214] In steps S1.11 to S1.16, the PCR reaction procedure is: ① pre-denaturation at 95°C for 3 min; ② denaturation at 95°C for 15 s; ③ annealing at 55°C for 15 s; ④ extension at 72°C for 1 min; ⑤ repeat ② to ④ for 35 cycles; ⑥ 72°C for 5 min.

[0215] 1.2 Construction of recombinant Sc-1

[0216] S1.21. Prepare competent yeast: Pick up Saccharomyces cerevisiae and inoculate into 5 mL of YPD liquid medium. Then, shake and culture at 30°C and 220 rpm for 12 h. Then, transfer the activated Saccharomyces cerevisiae to 50 mL of YPD liquid medium and adjust the starting OD 600 Adjust to 0.2 and culture at 30°C and 220 r / min until OD 600 is between 0.7 and 1.0 (the culture time is about 5 hours) to obtain a culture solution; the culture solution is centrifuged at 3000 r / min for 5 minutes, 50 mL of pure water is added to the bacteria from which the supernatant is discarded, the solution is centrifuged at 3000 r / min for 5 minutes, the supernatant is discarded, and then 20 mL of 100 mmol / L lithium acetate is added. After mixing, the solution is centrifuged again at 3000 r / min for 5 minutes, the supernatant is discarded again, and finally 400 μL of 100 mmol / L lithium acetate is added to resuspend the cells to obtain a competent state of Saccharomyces cerevisiae;

[0217] S1.22. Transform the target gene fragment into Saccharomyces cerevisiae competent cells: Take 30 μL of the Saccharomyces cerevisiae competent cells prepared in step S1.21 and place them in a 1.5 mL centrifuge tube. Add 240 μL of PEG3350 aqueous solution (50 g of PEG3350 is diluted to 100 mL with pure water), 36 μL of 1 mol / L lithium acetate, 5 μL of 10 mg / mL single-stranded fish sperm DNA (which needs to be boiled at 99°C for 5-10 minutes before use and then immediately placed on ice), 1 μg of the target gene fragment synthesized in step S1.17, 300 μL of PEG3350 solution, and 1 μL of 10 mg / mL single-stranded fish sperm DNA. ng of pCUT (511b-ura) plasmid and 70 μL of pure water were mixed to obtain a transformation system; the transformation system was first cultured at 30°C for 30 min, and then heat-shocked at 42°C for 40 min; the heat-shocked transformation solution was centrifuged at 5000 r / min for 1 min, the supernatant was discarded, 1 mL of water was added and mixed, and then centrifuged at 5000 r / min for 1 min, the supernatant was discarded again, and 100 μL of pure water was added and mixed to obtain a bacterial solution; the bacterial solution was evenly spread on an SC-URA medium plate and cultured at 30°C for 48 h;

[0218] S1.23. Verification: Pick a single clone on the transformation plate and culture it in SC-URA liquid medium for 12 hours, then take 200 μL of bacterial solution and centrifuge it at 5000 r / min for 1 minute, collect the supernatant, add 100 μL of 20 mmol / L NaOH aqueous solution to the supernatant, place it at 99°C for lysis for 20 minutes, and then immediately place it on ice for 5 minutes to obtain bacterial lysate; prepare a verification PCR system, which is shown in Table 4 below. The reaction procedure for verification PCR is: ① pre-denaturation at 94°C for 2 minutes; ② denaturation at 98°C for 10 seconds; ③ annealing at 55°C for 30 seconds; ④ extension at 72°C for 2 minutes; ⑤ repeat ② to ④ for 35 cycles; ⑥ 72°C for 5 minutes; electrophorese the PCR product with 1% agarose nucleic acid, and the target band size is 2339 bp. The PCR product with the correct fragment size is subjected to Sanger sequencing. The clone with the correct sequencing result is the recombinant Sc-1.

[0219] Table 4 Verification PCR reaction system list

[0220] The nucleotide sequence of the forward primer F7 is shown in SEQ ID NO.22, and the nucleotide sequence of the reverse primer R7 is shown in SEQ ID NO.23.

[0221] Example 2

[0222] This example provides a recombinant Sc-2 and a method for constructing the same. The recombinant Sc-2 is capable of expressing a fusion protein PLIN1-STP22. The fusion protein PLIN1-STP22 comprises a lipid droplet localization peptide and an STP22 protein in the ESCRT-I complex. The lipid droplet localization peptide has an amino acid sequence as shown in SEQ ID NO. 19, and the STP22 protein has an amino acid sequence as shown in SEQ ID NO. 24.

[0223] Compared with the construction method of recombinant Sc-1 in Example 1, the difference in the construction method of recombinant Sc-2 in this example is that: the "nucleotide sequence encoding Snf7 protein" in step S1.12 is replaced with "nucleotide sequence encoding STP22 protein", and the target gene fragment in step S1.17 is replaced with the nucleotide sequence shown in SEQ ID NO.25, and the target gene fragment includes the upstream homology arm sequence (511b-HoL) of the integration site 511b, the ADH2 promoter, the nucleotide sequence for encoding the localization peptide of the liquid lipid droplet, the nucleotide sequence for encoding the fusion junction, the nucleotide sequence encoding the STP22 protein, the CYC1 terminator and the upstream homology arm sequence (511b-HoR) of the integration site 511b, which are connected in series.

[0224] Example 3

[0225] This example provides a recombinant Sc-3 and a method for constructing the same. The recombinant Sc-3 is capable of expressing a fusion protein PLIN1-VPS25. The fusion protein PLIN1-VPS25 comprises a lipid droplet localization peptide and a VPS25 protein in the ESCRT-Ⅱ complex. The lipid droplet localization peptide has an amino acid sequence as shown in SEQ ID NO.19, and the VPS25 protein has an amino acid sequence as shown in SEQ ID NO.26.

[0226] Compared with the construction method of recombinant Sc-1 in Example 1, the difference in the construction method of recombinant Sc-3 in this example is that: the "nucleotide sequence encoding Snf7 protein" in step S1.12 is replaced with "nucleotide sequence encoding VPS25 protein", and the target gene fragment in step S1.17 is replaced with the nucleotide sequence shown in SEQ ID NO.27, and the target gene fragment includes the upstream homology arm sequence (511b-HoL) of the integration site 511b, the ADH2 promoter, the nucleotide sequence for encoding the localization peptide of the liquid lipid droplet, the nucleotide sequence for encoding the fusion junction, the nucleotide sequence encoding the VPS25 protein, the CYC1 terminator and the upstream homology arm sequence (511b-HoR) of the integration site 511b.

[0227] Example 4

[0228] This embodiment provides a recombinant Sc-4 and a method for constructing the same. The recombinant Sc-4 can express the fusion protein PLIN1-VTA1. The fusion protein PLIN1-VTA1 comprises a lipid droplet localization peptide and a VTA1 protein in the Vps4-VTA1 complex. The lipid droplet localization peptide has an amino acid sequence as shown in SEQ ID NO.19, and the VTA1 protein has an amino acid sequence as shown in SEQ ID NO.28.

[0229] Compared with the construction method of recombinant Sc-1 in Example 1, the difference in the construction method of recombinant Sc-4 in this example is that: the "nucleotide sequence encoding Snf7 protein" in step S1.12 is replaced with "nucleotide sequence encoding VTA1 protein", and the target gene fragment in step S1.17 is replaced with the nucleotide sequence shown in SEQ ID NO.29, and the target gene fragment includes the upstream homology arm sequence (511b-HoL) of the integration site 511b, the ADH2 promoter, the nucleotide sequence for encoding the localization peptide of the liquid lipid droplet, the nucleotide sequence for encoding the fusion junction, the nucleotide sequence encoding the VTA1 protein, the CYC1 terminator and the upstream homology arm sequence (511b-HoR) of the integration site 511b, which are connected in series.

[0230] Comparative Example 1

[0231] This comparative example provides a recombinant Sc-5 and a construction method thereof. The recombinant Sc-5 can overexpress a PLIN1 lipid droplet localization peptide, wherein the lipid droplet localization peptide has an amino acid sequence shown in SEQ ID NO.19.

[0232] Compared with the construction method of recombinant Sc-1 in Example 1, the difference in the construction method of recombinant Sc-5 in this comparative example is that: the second gene fragment in step S1.12 is replaced by "the second gene fragment contains a homologous sequence of a nucleotide sequence for encoding a localization peptide of liquid lipid droplets and a CYC1 terminator homologous sequence connected in series", and the target gene fragment in step S1.17 is replaced by "consisting of the upstream homologous arm sequence (511b-HoL) of the integration site 511b, the ADH2 promoter, the nucleotide sequence for encoding the localization peptide of liquid lipid droplets, the CYC1 terminator and the upstream homologous arm sequence (511b-HoR) of the integration site 511b connected in series".

[0233] Comparative Example 2

[0234] This comparative example provides a recombinant Sc-6 and a construction method thereof. The recombinant Sc-6 can overexpress the Snf7 protein in the ESCRT-III complex. The Snf7 protein has the amino acid sequence shown in SEQ ID NO.20.

[0235] Compared with the construction method of recombinant Sc-1 in Example 1, the difference in the construction method of recombinant Sc-6 in this comparative example is that the second gene fragment in step S1.12 is replaced by "the second gene fragment contains a nucleotide sequence encoding Snf7 protein and a CYC1 terminator homologous sequence connected in series", and the target gene fragment in step S1.17 is replaced by "consisting of the upstream homologous arm sequence (511b-HoL) of the integration site 511b, the ADH2 promoter, the nucleotide sequence encoding Snf7 protein, the CYC1 terminator and the upstream homologous arm sequence (511b-HoR) of the integration site 511b connected in series".

[0236] Experimental example

[0237] The recombinant strains of Examples 1 to 4, Comparative Examples 1 to 4, and Saccharomyces cerevisiae YB / I / E extra I (as a control) were fermented under the same conditions to produce β-carotene, and the differences in the extracellular yield of β-carotene produced by the various recombinant strains were compared.

[0238] The fermentation culture of each recombinant comprises the following steps:

[0239] S101. Inoculate the recombinant into 5 mL of YPD liquid medium and culture at 30°C and 220 rpm until the OD 600 The value of is 0.5, and the first culture solution is obtained;

[0240] S102, covering the surface of the culture solution in step S101 with a layer of dodecane (the volume ratio of dodecane to culture solution is 1:10), and continuing to culture at 30°C and 220 rpm for 6 days to obtain a second culture solution;

[0241] S103, centrifuging the second culture solution at 4000 rpm for 10 min, and collecting the supernatant, which contains β-carotene;

[0242] S104. Detect the content of β-carotene in the supernatant obtained in step S103 using HPLC technology.

[0243] The extracellular yields of β-carotene produced by each recombinant fermentation are shown in Table 1 below:

[0244] Table 1 Summary of extracellular yields of β-carotene produced by various recombinant fermentations

[0245] As shown in Table 1, compared with the extracellular yield of β-carotene produced by the recombinants of Comparative Examples 1 and 2 and the fermentation of Saccharomyces cerevisiae YB / I / E extra I, the extracellular yield of β-carotene produced by the recombinants in Examples 1 to 4 is higher. Taking the Sc-1 recombinant and Saccharomyces cerevisiae YB / I / E extra I in Example 1 as an example, as shown in Figures 3 and 4, the extracellular yield of β-carotene produced by the Sc-1 recombinant fermentation is 9 times the extracellular yield of β-carotene produced by Saccharomyces cerevisiae YB / I / E extra I fermentation. It can be seen that for the recombinant capable of expressing a fusion protein (comprising a lipid droplet localization peptide and an ESCRT protein), the fusion protein can be localized on the surface of the lipid droplets in the recombinant, thereby entering the sorting link of the lipid droplets storing β-carotene through the ESCRT pathway, and finally entering the exosomes, and secreted to the extracellular space through the exosomes, effectively increasing the extracellular yield of β-carotene.

[0246] From the extracellular yield data of β-carotene produced by recombinant fermentation in Examples 1 to 4, Comparative Examples 1 and 2, it can be seen that compared with the recombinant capable of expressing the localization peptide or ESCRT protein that produces liquid lipid droplets, the recombinant capable of expressing the fusion protein (comprising the localization peptide of liquid lipid droplets and the ESCRT protein) has a higher extracellular yield of β-carotene produced by fermentation. Therefore, only overexpressing the localization peptide of liquid lipid droplets or overexpressing the ESCRT protein cannot effectively increase the extracellular yield of β-carotene.

[0247] In the above embodiments, the description of each embodiment has its own focus. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0248] The above is a detailed introduction to a fusion protein provided in the examples of the present application and its application in the preparation of metabolites. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above examples is only used to help understand the method of the present application and its core idea. At the same time, for those skilled in the art, according to the ideas of the present application, there may be changes in the specific implementation methods and application scopes. In summary, the contents of this specification should not be understood as limiting the present application.

Claims

1. A fusion protein, wherein: The fusion protein comprises a localization peptide of the first subcellular structure and an ESCRT protein, wherein the ESCRT protein comprises one or more of the following (a) to (f): (a) one or more proteins in the ESCRT-0 complex; (b) one or more proteins in the ESCRT-I complex; (c) one or more proteins in the ESCRT-II complex; (d) one or more proteins in the ESCRT-III complex; (e) one or more proteins in the Vps4-Vta1 complex; (f) One or more proteins in the ALIX dimer.

2. The fusion protein according to claim 1, wherein The one or more proteins in the ESCRT-0 complex are selected from one or more of Vps27, Hse1, HRS, STAM1 and STAM2; The one or more proteins in the ESCRT-I complex are selected from one or more of Vps23, Vps28, Vps37, Mvb12, STP22, TSG101, VPS28, VPS37A, VPS37B, VPS37C, VPS37D, MVB12A, MVB12B, UBAP1, UBA1L and UMAD1; The one or more proteins in the ESCRT-Ⅱ complex are selected from one or more of Vps22, Vps25, Vps36, EPA30, EPA20 and EPA45; The one or more proteins in the ESCRT-III complex are selected from one or more of Vps2, Vps20, Snf7, Vps24, Did2, Vps60, Ist1, CHMP2A, CHMP2B, CHMP6, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP5, CHMP1A, CHMP1B, CHMP7 and IST1; The one or more proteins in the Vps4-Vta1 complex are selected from one or more of Vta1, Vps4, SKD1, SKD2 and LIP5; The one or more proteins in the ALIX dimer are selected from one or more of Brol, ALIX and HD-PTP.

3. The fusion protein according to claim 1, wherein The first subcellular structure is selected from one or more of lipid droplets, peroxisomes, lysosomes, mitochondria, centrosomes, Golgi bodies and microsomes.

4. The fusion protein according to claim 1, wherein The fusion protein further comprises a fusion linker for connecting the localization peptide and the ESCRT protein.

5. The fusion protein according to claim 1, wherein The first subcellular structure is a lipid droplet, and the localization peptide of the first subcellular structure is one or more of the following (A1) to (A3): (A1) has the amino acid sequence shown in SEQ ID NO.19; (A2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO.19; (A3) The amino acid sequence is obtained by replacing at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid in the amino acid sequence shown in SEQ ID NO. 19, and has the function of localizing on the surface of the lipid droplet.

6. The fusion protein according to claim 5, wherein The second protein is Snf7, and the Snf7 is one or more of the following (B1) to (B3): (B1) has the amino acid sequence shown in SEQ ID NO.20; (B2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO.20; (B3) The amino acid sequence is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid in the amino acid sequence shown in SEQ ID NO.20, and has the same function as the protein of the amino acid sequence shown in SEQ ID NO.

20.

7. The fusion protein according to claim 6, wherein The fusion protein is one or more of the following (C1) to (C3): (C1) has the amino acid sequence shown in SEQ ID NO.21; (C2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO.21; The (C3) amino acid sequence is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid in the amino acid sequence shown in SEQ ID NO.21, and has the same function as the protein of the amino acid sequence shown in SEQ ID NO.

21.

8. The fusion protein according to claim 5, wherein The second protein is STP22, and the STP22 is one or more of the following (D1) to (D3): (D1) has the amino acid sequence shown in SEQ ID NO.24; (D2) The amino acid sequence has no less than 80% similarity to the amino acid sequence shown in SEQ ID NO.24 Homology; The (D3) amino acid sequence is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid in the amino acid sequence shown in SEQ ID NO.24, and has the same function as the protein of the amino acid sequence shown in SEQ ID NO.

24.

9. The fusion protein according to claim 5, wherein The second protein is VPS25, and the VPS25 is one or more of the following (E1) to (E3): (E1) has the amino acid sequence shown in SEQ ID NO.26; (E2) an amino acid sequence having no less than 80% homology with the amino acid sequence shown in SEQ ID NO.26; (E3) The amino acid sequence is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid in the amino acid sequence shown in SEQ ID NO.26, and has the same function as the protein of the amino acid sequence shown in SEQ ID NO.

26.

10. The fusion protein according to claim 5, wherein The second protein is VTA1, and the VTA1 is one or more of the following (F1) to (F3): (F1) has the amino acid sequence shown in SEQ ID NO.28; (F2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO.28; The (F3) amino acid sequence is obtained by substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid in the amino acid sequence shown in SEQ ID NO.28, and has the same function as the protein of the amino acid sequence shown in SEQ ID NO.

28.

11. A nucleic acid molecule, wherein The nucleic acid molecule comprises a nucleotide sequence encoding a fusion protein, wherein the fusion protein comprises a localization peptide of a first subcellular structure and an ESCRT protein, wherein the ESCRT protein comprises one or more of the following (a) to (f): (a) one or more proteins in the ESCRT-0 complex; (b) one or more proteins in the ESCRT-I complex; (c) one or more proteins in the ESCRT-II complex; (d) one or more proteins in the ESCRT-III complex; (e) one or more proteins in the Vps4-Vta1 complex; (f) One or more proteins in the ALIX dimer.

12. The nucleic acid molecule according to claim 11, wherein The nucleic acid molecule is one or more of the following (G1) to (G3): (G1) has the nucleotide sequence shown in SEQ ID NO.30; (G2) the nucleotide sequence has no less than 80% homology with the nucleotide sequence shown in SEQ ID NO.30; The (G3) nucleotide sequence is obtained by replacing at least one nucleotide, and / or deleting at least one nucleotide, and / or adding at least one nucleotide of the nucleotide sequence shown in SEQ ID NO.30, and has the function of encoding the fusion protein.

13. The nucleic acid molecule according to claim 11, wherein The nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO.25, or the nucleotide sequence shown in SEQ ID NO.27, or the nucleotide sequence shown in SEQ ID NO.

29.

14. A recombinant expression vector, wherein: The invention comprises a vector and a nucleic acid molecule loaded in the vector, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a fusion protein, wherein the fusion protein comprises a localization peptide of a first subcellular structure and an ESCRT protein, wherein the ESCRT protein comprises one or more of the following (a) to (f): (a) one or more proteins in the ESCRT-0 complex; (b) one or more proteins in the ESCRT-I complex; (c) one or more proteins in the ESCRT-II complex; (d) one or more proteins in the ESCRT-III complex; (e) one or more proteins in the Vps4-Vta1 complex; (f) One or more proteins in the ALIX dimer.

15. The recombinant expression vector according to claim 14, wherein The vector is selected from the group consisting of a plasmid, a phage, a phagemid, a cosmid, a virus, a yeast artificial chromosome or a bacterial artificial chromosome.

16. A recombinant, wherein The recombinant comprises a host, and a nucleic acid molecule or a recombinant expression vector introduced into the host, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a fusion protein or the recombinant expression vector comprises a nucleotide sequence encoding a fusion protein, wherein the fusion protein comprises a localization peptide of a first subcellular structure and an ESCRT protein, wherein the ESCRT protein comprises one or more of the following (a) to (f): (a) one or more proteins in the ESCRT-0 complex; (b) one or more proteins in the ESCRT-I complex; (c) one or more proteins in the ESCRT-II complex; (d) one or more proteins in the ESCRT-III complex; (e) one or more proteins in the Vps4-Vta1 complex; (f) One or more proteins in the ALIX dimer.

17. The recombinant according to claim 16, wherein The host is yeast.

18. A method for preparing a fusion protein, wherein: The recombinant according to claim 16 is fermented and cultured to express the fusion protein.

19. The preparation method according to claim 18, wherein: The method for introducing the nucleic acid molecule into the host comprises the following steps: Providing the nucleic acid molecule, the nucleic acid molecule comprises a first nucleotide sequence to a third nucleotide sequence connected in series, wherein the first nucleotide sequence is an upstream homology arm sequence of the integration site in the host genome, the second nucleotide sequence is a nucleotide sequence encoding the fusion protein, and the third nucleotide sequence is a downstream homology arm sequence of the integration site in the host genome; and The nucleic acid molecule is transformed into the host, and homologous recombination occurs between the nucleic acid molecule and the genome of the host.

20. A method for preparing a metabolite, wherein: The method comprises the steps of: fermenting and culturing the recombinant as claimed in claim 16 to express and produce the fusion protein, thereby secreting the second subcellular structure storing the target metabolite to the extracellular space through the ESCRT pathway; wherein the first subcellular structure is the same as the second subcellular structure.

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