Fusion protein and application thereof in preparation of metabolites

Abstract

The invention discloses a fusion protein and application thereof in preparation of metabolites, the fusion protein comprises a positioning peptide of a first subcellular structure and an ESCRT protein, the fusion protein can be applied to preparation of the metabolites, and for a host capable of expressing the fusion protein, in the host body, the ESCRT protein has a first subcellular structure and a second subcellular structure. The fusion protein can be positioned on the surface of the second subcellular structure which is the same as the first subcellular structure, so that the second subcellular structure stored with the target metabolite enters a sorting link through an ESCRT path, finally enters the exosome and is secreted to the outside of the cell through the exosome, the extracellular yield of the target metabolite is effectively increased, and the target metabolite can be used for screening the target metabolite. And a cell breaking process can be omitted, so that the synthesis cost of the target metabolite is reduced, and the purity of the target metabolite is improved.

Description

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 variety of metabolites during the metabolic process. Based on the relationship between metabolites and microbial growth and reproduction, microbial metabolites can be divided into two categories: primary metabolites and secondary metabolites. Primary metabolites refer to substances produced by microorganisms through metabolic activities and necessary for their own growth and reproduction. Secondary metabolites refer to substances that are produced when microorganisms grow to a certain stage and have a very complex chemical structure, have no obvious physiological function for the microorganism, or are not necessary for the growth and reproduction of microorganisms. Secondary metabolites include but are not limited to terpenoids, alkaloids and flavonoids.

[0003] Secondary metabolites synthesized by microorganisms usually exist inside cells. In order to obtain secondary metabolites, cell disruption, separation and purification are often required, which has the disadvantages of being time-consuming and labor-intensive, with high extraction costs and reduced product purity. Therefore, how to provide a protein that can promote the secretion of target secondary metabolites outside cells is of great significance to simplifying the production process of secondary metabolites and reducing the production cost of secondary metabolites, and is conducive to promoting the application and development of secondary metabolites. Summary of the invention

[0004] 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 is applied to the preparation of target secondary metabolites.

[0005] The technical solution of this application is as follows:

[0006] In a first aspect, the present application provides 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):

[0007] (a) one or more proteins in the ESCRT-0 complex;

[0008] (b) one or more proteins in the ESCRT-I complex;

[0009] (c) one or more proteins in the ESCRT-II complex;

[0010] (d) one or more proteins in the ESCRT-III complex;

[0011] (e) one or more proteins in the Vps4-Vta1 complex;

[0012] (f) One or more proteins in the ALIX dimer.

[0013] Optionally, 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

[0014] 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

[0015] The one or more proteins in the ESCRT-Ⅱ complex are selected from one or more of Vps22, Vps25, Vps36, EPA30, EPA20 and EPA45; and / or

[0016] 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

[0017] The one or more proteins in the Vps4-Vta1 complex are selected from one or more of Vta1, Vps4, SKD1, SKD2 and LIP5; and / or

[0018] The one or more proteins in the ALIX dimer are selected from one or more of Brol, ALIX and HD-PTP.

[0019] Optionally, the first subcellular structure is selected from one or more of lipid droplets, peroxisomes, lysosomes, mitochondria, centrosomes, Golgi bodies and microsomes; and / or

[0020] 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 respectively.

[0021] 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):

[0022] (A1) has the amino acid sequence shown in SEQ ID NO.19;

[0023] (A2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO.19;

[0024] (A3) 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 from the amino acid sequence shown in SEQ ID NO.19, and has the function of localizing on the surface of the lipid droplet;

[0025] And / or, the second protein is Snf7, and the Snf7 is one or more of the following (B1) to (B3):

[0026] (B1) has the amino acid sequence shown in SEQ ID NO.20;

[0027] (B2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO.20;

[0028] (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 of the amino acid sequence shown in SEQ ID NO.20;

[0029] And / or, the fusion protein is one or more of the following (C1) to (C3):

[0030] (C1) has the amino acid sequence shown in SEQ ID NO.21;

[0031] (C2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO.21;

[0032] 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.

[0033] In a second aspect, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding a fusion protein as described in any one of the first aspects;

[0034] Optionally, the nucleic acid molecule further comprises one or more of a promoter, an intron, an exon, a terminator and an enhancer; and / or, the nucleic acid molecule is one or more of the following (D1) to (D3):

[0035] (G1) has the nucleotide sequence shown in SEQ ID NO.30;

[0036] (G2) the nucleotide sequence has no less than 80% homology with the nucleotide sequence shown in SEQ ID NO.30;

[0037] 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.

[0038] In a third aspect, the present application provides a recombinant expression vector, comprising a vector and a nucleic acid molecule as described in any one of the second aspects, wherein the nucleic acid molecule is loaded in the vector;

[0039] 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.

[0040] In a fourth aspect, the present application provides a recombinant, comprising a host, and a nucleic acid molecule as described in any one of the second aspects, or a recombinant expression vector as described in any one of the third aspects, introduced into the body of the host;

[0041] Optionally, the host is yeast.

[0042] In a fifth aspect, the present application provides a method for preparing a fusion protein, comprising the steps of: fermenting and culturing the recombinant as described in any one of the fourth aspects to express and obtain the fusion protein.

[0043] Optionally, the method for introducing the nucleic acid molecule into the host as described in any one of the second aspects comprises the following steps:

[0044] 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 genome of the host, the second nucleotide sequence is a nucleotide sequence encoding the fusion protein as described in any one of the first aspects, and the third nucleotide sequence is a downstream homology arm sequence of the integration site in the genome of the host; and

[0045] The nucleic acid molecule is transformed into the host, and homologous recombination occurs between the nucleic acid molecule and the genome of the host.

[0046] In a sixth aspect, the present application provides a method for preparing a metabolite, comprising the steps of: fermenting and culturing the recombinant as 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.

[0047] The present application provides a fusion protein and its application in preparing metabolites, which has the following technical effects:

[0048] In the fusion protein of the present application, the fusion protein comprises a localization peptide of a first subcellular structure and an ESCRT protein. The fusion protein can be used to prepare metabolites. For a host capable of expressing the fusion protein, in the host body, the fusion protein can be localized on the surface of a second subcellular structure that is identical to the first 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 outside the cell through the exosomes, thereby effectively increasing the extracellular yield of the target metabolite, and omitting the cell disruption step, 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

[0049] The technical solution and beneficial effects of the present application will be made apparent by describing in detail the specific implementation modes of the present application in conjunction with the accompanying drawings.

[0050] Figure 1 This is a schematic diagram of the composition of the fusion protein provided in the examples of this application;

[0051] Figure 2 This is a schematic diagram of the composition of the target gene fragment provided in the examples of this application;

[0052] Figure 3 This is a graph showing the extracellular production of β-carotene by each recombinant and control fermentation in the experimental examples of this application;

[0053] Figure 4 This is a comparison picture of the supernatant of the Sc-1 recombinant and the control in the experimental example of this application. DETAILED DESCRIPTION

[0054] The terms used in this application are defined as follows:

[0055] 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 multivesicular 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 system of humans and yeast has a high homology, and the domains or motifs that play their specific roles are similar.

[0056] As used in this application, "plurality" means two or more, for example, two, three, four, five, six, seven, eight, nine, ten, etc.

[0057] 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 arbitrary and all combinations include any combination of two related listed items, any more related listed items, or 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, all technical solutions connected by "logical or"), and also includes any and all combinations of A, B, C, and D, that is, including any combination of any two or any three of A, B, C, and D, and also includes a combination of four items of A, B, C, and D (that is, all technical solutions connected by "logical and").

[0058] 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 the identical nucleotides or amino acid residues between the two sequences to be compared obtained after the 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 the identical nucleotides or amino acid residues between the two sequences to be compared obtained after the 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 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).

[0059] 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, exemplified by 80%, 81%, 82%, 83%, 84%, , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology, and 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 less than 100% sequence homology, the corresponding homology value is a decimal.

[0060] As used in this application, "nucleic acid molecule" refers to a biomacromolecule compound containing 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, which 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.

[0061] As used in this application, "recombinant expression vector" refers to a nucleic acid molecule construct containing an exogenous nucleic acid fragment operably linked to a suitable control sequence, which can achieve the 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) structures or coding sequences that are transcribed into mRNA and translated into proteins. 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.

[0062] As used in this application, "vector" refers to a tool that uses genetic engineering recombination technology to transfer target nucleic acid molecules to 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 can use appropriate vectors to implement the technical solutions of this application, but these substitutions do not deviate from the scope of this application, and this application should include these alternative vectors.

[0063] As used in this application, "host" refers to a type of recipient microorganism that can accept exogenous nucleic acid (such as the nucleic acid molecule of the present application) in a specific manner, and the specific manner includes but is not limited to at least one of conjugation, electroporation, chemical transformation, transduction, transfection or ultrasonic transformation.

[0064] This application studies a fusion protein and its application in preparing metabolites, such as Figure 1As shown, the fusion protein comprises a localization peptide of a first subcellular structure and an ESCRT protein, and the ESCRT protein comprises one or more of the following (a) to (f):

[0065] (a) one or more proteins in the ESCRT-0 complex;

[0066] (b) one or more proteins in the ESCRT-I complex;

[0067] (c) one or more proteins in the ESCRT-II complex;

[0068] (d) one or more proteins in the ESCRT-III complex;

[0069] (e) one or more proteins in the Vps4-Vta1 complex;

[0070] (f) One or more proteins in the ALIX dimer.

[0071] Among them, the structural composition of each complex in the ESCRT system is shown in Table 1 below:

[0072] Table 1 Structural composition of each complex in the ESCRT system

[0073]

[0074] 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.

[0075] 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 of the proteins in 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.

[0076] 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, and the two or more proteins can be connected in series through a linker. As an example, the ESCRT protein in the fusion protein contains two proteins, both of which can be proteins in the ESCRT-III complex, or the two proteins can be a protein in the ESCRT-I complex and a protein in the ESCRT-II complex. As an example, the ESCRT protein in the fusion protein contains three proteins, all of which can be proteins in the ESCRT-III complex, or the three proteins can be a protein in the ESCRT-I complex, a protein in the ESCRT-II complex, and a protein in the ESCRT-III complex.

[0077] 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.

[0078] In some embodiments of the present application, see Figure 1The fusion protein also includes a fusion linker for connecting the localization peptide of the first subcellular structure with the ESCRT protein, and a fusion linker commonly used in the art can be used. 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.

[0079] It should be noted that in the fusion protein, the connection order of the localization peptide of the first subcellular structure and the ESCRT protein is not specifically limited, and the localization peptide of the first subcellular structure may be closer to the N-terminus or the C-terminus. When the ESCRT protein comprises multiple proteins, the connection order of the multiple proteins is also not specifically limited.

[0080] 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.

[0081] 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):

[0082] (A1) has the amino acid sequence shown in SEQ ID NO.19;

[0083] (A2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO.19;

[0084] (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.

[0085] For (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, and the variant form has the function of localizing on the surface of the liquid lipid droplet, and there is a small difference from the amino acid sequence shown in SEQ ID NO.19, and 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 deletion, insertion and / or substitution, and adding one or more (usually within 20, such as within 10, 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 used for substitution, the function of the protein is usually not changed; in addition, adding one or more amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein.

[0086] In some embodiments of the present application, the second protein is Snf7, and Snf7 is one or more of the following (B1) to (B3):

[0087] (B1) has the amino acid sequence shown in SEQ ID NO.20;

[0088] (B2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO.20;

[0089] (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.

[0090] Regarding (B3), it should be noted that Snf7 can be a variant form of the amino acid sequence shown in SEQ ID NO.20, and the variant form has the same or similar function as Snf7, and has a small difference from the amino acid sequence shown in SEQ ID NO.20, and these variant forms include but are not limited to: one or more (usually 1 to 50, for example 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 deletion, insertion and / or substitution, and addition of one or more (usually within 20, for example within 10, such as within 6) amino acids at the C-terminus and / or N-terminus.

[0091] Furthermore, the fusion protein is one or more of the following (C1) to (C3):

[0092] (C1) has the amino acid sequence shown in SEQ ID NO.21;

[0093] (C2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO.21;

[0094] 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.

[0095] It should be noted that (C3) the fusion protein may be a variant form of the amino acid sequence shown in SEQ ID NO.21, and there is 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: 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, such as 1 to 6) amino acid deletions, insertions and / or substitutions, and one or more (usually within 20, such as within 10, such as within 6) amino acids added to the C-terminus and / or N-terminus.

[0096] 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):

[0097] (D1) has the amino acid sequence shown in SEQ ID NO.24;

[0098] (D2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO.24;

[0099] 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.

[0100] Regarding (D3), it should be noted that STP22 can 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, and these variant forms include but are not limited to: one or more (usually 1 to 50, for example 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 deletion, insertion and / or substitution, and addition of one or more (usually within 20, for example within 10, such as within 6) amino acids at the C-terminus and / or N-terminus.

[0101] 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):

[0102] (E1) has the amino acid sequence shown in SEQ ID NO.26;

[0103] (E2) an amino acid sequence having no less than 80% homology with the amino acid sequence shown in SEQ ID NO.26;

[0104] (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.

[0105] Regarding (E3), it should be noted that VPS25 can be a variant form of the amino acid sequence shown in SEQ ID NO.26, and the variant form 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, such as 1 to 20, such as 1 to 10, such as 1 to 8, and such as 1 to 6) amino acid deletion, insertion and / or substitution, and addition of one or more (usually within 20, for example within 10, such as within 6) amino acids at the C-terminus and / or N-terminus.

[0106] 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):

[0107] (F1) has the amino acid sequence shown in SEQ ID NO.28;

[0108] (F2) the amino acid sequence has no less than 80% homology with the amino acid sequence shown in SEQ ID NO.28;

[0109] 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.

[0110] Regarding (F3), it should be noted that VTA1 can be a variant form of the amino acid sequence shown in SEQ ID NO.28, and the variant form has the same or similar function as VTA1, and has a small difference from the amino acid sequence shown in SEQ ID NO.28, and these variant forms include but are not limited to: one or more (usually 1 to 50, for example 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 deletion, insertion and / or substitution, and addition of one or more (usually within 20, for example within 10, and such as within 6) amino acids at the C-terminus and / or N-terminus.

[0111] 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, in the host body, the fusion protein can be localized on the surface of a second subcellular structure that is identical to the first 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 outside the cell through the exosomes, thereby effectively increasing the extracellular yield of the target metabolite, and omitting the cell disruption step, which is beneficial to reducing the synthesis cost of the target metabolite and improving the purity of the target metabolite.

[0112] The embodiments of the present application also provide a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence encoding any one of the fusion proteins described above.

[0113] In some embodiments of the present application, the nucleic acid molecule is one or more of the following (G1) to (G3):

[0114] (G1) has the nucleotide sequence shown in SEQ ID NO.30;

[0115] (G2) the nucleotide sequence has no less than 80% homology with the nucleotide sequence shown in SEQ ID NO.30;

[0116] 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.

[0117] It should be noted that for (G3), 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 a substitution variant, a deletion variant and an insertion variant. As is known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the protein encoded by it.

[0118] In some embodiments of the present application, the nucleic acid molecule is one or more of the following (H1) to (H3):

[0119] (H1) has the nucleotide sequence shown in SEQ ID NO.25;

[0120] (H2) the nucleotide sequence has no less than 80% homology with the nucleotide sequence shown in SEQ ID NO.25;

[0121] (H3) 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 of the nucleotide sequence shown in SEQ ID NO. 25, and has the function of encoding the fusion protein.

[0122] It should be noted that for (H3), the nucleic acid molecule may be a variant of a polynucleotide having a nucleotide sequence as shown in SEQ ID NO.25, and has the function of encoding a fusion protein PLIN1-STP22. The variant may be a naturally occurring allelic variant or a non-naturally occurring variant, including but not limited to a substitution variant, a deletion variant and an insertion variant. As is known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the protein encoded by it.

[0123] In some embodiments of the present application, the nucleic acid molecule is one or more of the following (I1) to (I3):

[0124] (I1) having the nucleotide sequence shown in SEQ ID NO.27;

[0125] (I2) the nucleotide sequence has no less than 80% homology with the nucleotide sequence shown in SEQ ID NO.27;

[0126] (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 of the nucleotide sequence shown in SEQ ID NO. 27, and has the function of encoding the fusion protein.

[0127] It should be noted that for (I3), the nucleic acid molecule may be a variant of a polynucleotide having a nucleotide sequence as shown in SEQ ID NO.27, and has 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 a substitution variant, a deletion variant and an insertion variant. As is known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the protein encoded by it.

[0128] In some embodiments of the present application, the nucleic acid molecule is one or more of the following (J1) to (J3):

[0129] (J1) having the nucleotide sequence shown in SEQ ID NO.29;

[0130] (J2) the nucleotide sequence has no less than 80% homology with the nucleotide sequence shown in SEQ ID NO.29;

[0131] (J3) 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 of the nucleotide sequence shown in SEQ ID NO. 29, and has the function of encoding the fusion protein.

[0132] It should be noted that for (J3), the nucleic acid molecule may be a variant of a polynucleotide having a nucleotide sequence as shown in SEQ ID NO.29, and has the function of encoding 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 a substitution variant, a deletion variant and an insertion variant. As is known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the protein encoded by it.

[0133] It should be noted that the nucleic acid molecule, for example, 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 of 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, wherein 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 series order only brings about a difference in operation and does not affect the expression effect.

[0134] The embodiment of the present application also provides a recombinant expression vector, comprising a vector and any one of the nucleic acid molecules described above, wherein the nucleic acid molecule is loaded in the vector.

[0135] In the recombinant expression vector, the nucleic acid molecule may be a single copy or multiple copies, for example, the nucleic acid molecule may be a double copy. When the nucleic acid molecule is multiple copies, they may be sequentially connected end to end to form a tandem repeat sequence.

[0136] The embodiments of the present application also provide a recombinant, which comprises a host, and a nucleic acid molecule as described in any one of the foregoing, or a recombinant expression vector as described in any one of the foregoing, introduced into the host.

[0137] 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 fermentation, Saccharomyces paradoxus, Saccharomyces vitis, Saccharomyces bayanus, Schizosaccharomyces, Kluyveromyces, Torulospora, Pichia, Zygosaccharomyces, Brettanomyces or Metschnis. Among them, the genus Schizosaccharomyces includes but is not limited to Schizosaccharomyces pombe, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus or Psychrophilic Schizosaccharomyces; the genus Torulospora includes but is not limited to Saccharomyces de Boer; 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 brusselsii, Brettanomyces heterotypicus, Brettanomyces bantu, Brettanomyces nadsii, Brettanomyces nanus, Dekkera brusselsii or Dekkera heterotypicus; the genus Metschnis includes but is not limited to Issatchenia or Kloeckneria. As an example, the host is Saccharomyces cerevisiae.

[0138] It should be noted that the host can be a wild type or a microorganism obtained after natural mutation or artificial modification of the wild type. In the embodiment of the present application, the host is Saccharomyces cerevisiae YB / I / E extra I, which is a recombinant strain with high β-carotene production obtained after artificial modification.

[0139] The present application also provides a method for preparing a fusion protein, comprising the steps of: fermenting and culturing any of the above-mentioned recombinants to express and obtain the fusion protein. According to the selected host, the culture medium used in the fermentation culture can be selected from various conventional culture media, and cultured under conditions suitable for the growth of the host. Taking the host as Saccharomyces cerevisiae as an example, the culture medium can be YPD, the culture temperature can be 30°C, and the culture can be aerobic.

[0140] The fusion protein can be expressed in the host cell, or on the cell membrane, or secreted outside the cell. 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, including but not limited to: conventional renaturation treatment, treatment with protein precipitants (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 of these methods.

[0141] In some embodiments of the present application, the method of introducing any of the above-mentioned nucleic acid molecules into the body of a host comprises the following steps:

[0142] S1. Provide 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's 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's genome;

[0143] S2, transforming the nucleic acid molecule of step S1 into a host, and homologous recombination occurs between the nucleic acid molecule and the host genome.

[0144] In step S1, when the host is Saccharomyces cerevisiae, the integration site may be, for example, 511b.

[0145] In step S2, "transformation" can be performed using conventional techniques known to those skilled in the art, and an appropriate transformation method is selected according to the selected host. For example, when the host is Saccharomyces cerevisiae, the lithium acetate transformation method can be selected. In order to facilitate the screening of positive clones, the nucleic acid molecule can be transformed into the host together with the CRISPR-Cas system, and the CRISPR-Cas system can be used as a screening system to improve the problem of difficult screening in recombinant engineering.

[0146] 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 a 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.

[0147] It should be noted that microorganisms are often used for efficient heterologous synthesis of natural products (mainly metabolites of microorganisms). However, the synthesized natural products are stored in cells (e.g., in subcellular structures) and are not secreted outside the cells or are secreted in very small amounts outside the cells. They often require cell rupture to obtain. Taking lipid droplets as an example, lipid droplets are mainly composed of neutral fats and a small amount of phospholipids, triglycerides, proteins and other biologically active substances. They play a certain role in energy metabolism, lipid metabolism, synthesis and transport of cell membranes, material storage, and delivery of biologically active molecules. Lipid droplets are storage sites for fat-soluble compounds, including but not limited to carotenoids, withanolides, ubiquinones, polyterpenoids, sterols and biofuels (e.g., squalene).

[0148] 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 the same as 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 outside of the cell through the exosomes, thereby effectively increasing the extracellular yield of the target metabolite, and omitting the cell disruption step, which is beneficial to reducing the synthesis cost of the target metabolite and improving the purity of the target metabolite.

[0149] 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.

[0150] The technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying 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 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 the present application. The experimental methods in the following embodiments that do not specify specific conditions are usually carried out according to conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0151] 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 embodiments and comparative examples of the present application are commercially available. In addition, any method and material similar to or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only, but cannot limit the content of the present application. The terms "first", "second", "third" or similar expressions thereof are only used for descriptive purposes and cannot be interpreted as indicating or implying relative importance, nor as indicating or implying a sequence.

[0152] Ⅰ. Description of reagents and materials involved in the examples of this application:

[0153] The primers and gene fragments involved in the examples of this application were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0154] The PCR reaction reagents involved in the examples of the present application were purchased from Nanjing Novozyme Biotechnology Co., Ltd., among which the model of 2×Phanta Max Master Mix was P515;

[0155] 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 successful transformation with carotenogenic genes from Xanthophyllomyces dendrorhous. Appl Environ Microbiol, 73(13), 4342-4350. https: / / doi.org / 10.1128 / AEM.02759-06.

[0156] The construction method of the pCUT (511b-ura) plasmid involved in the examples of the present application is derived from the literature 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.

[0157] (https: / / doi.org / 10.1093 / nar / gkw1023).

[0158] The single-stranded fish sperm DNA involved in the examples of this application was purchased from Beijing Solebow Technology Co., Ltd., model number H1060.

[0159] 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.

[0160] The SC-URA culture medium involved in the examples of the present application was purchased from Beijing Coolbo Technology Co., Ltd., model number PM2273.

[0161] II. The specific information of the primers involved in the examples of this application is shown in Table 2 below:

[0162] Table 2 Primer list

[0163]

[0164] III. Detection method of β-carotene in the supernatant involved in the experimental examples of this application

[0165] The present application example uses high performance liquid chromatography (HPLC) to detect the content of β-carotene in the supernatant, uses the external standard method to quantify by peak area, and uses β-carotene standard (purchased from Sigma) as the qualitative and quantitative standard of β-carotene in the supernatant. The working conditions of HPLC are as follows:

[0166] (1) Preparation of injection solution: Filter the supernatant with a 0.22 μm filter. The filtered supernatant is the injection solution. 5 μL is injected each time.

[0167] (2) Chromatographic column: YMC Carotenoid column, 4.6 mm × 250 mm, 5 μm;

[0168] (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)

[0169] (4) Flow rate: 1 mL / min;

[0170] (6) Column temperature: 30°C;

[0171] (7) UV detection wavelength: 450nm.

[0172] The technical scheme and beneficial effects of the present invention are further illustrated below in conjunction with embodiments, comparative examples and experimental examples.

[0173] Example 1

[0174] This embodiment provides a recombinant Sc-1 and a construction method thereof. The recombinant Sc-1 can express a fusion protein PLIN1-Snf7, and the fusion protein PLIN1-Snf7 has the amino acid sequence shown in SEQ ID NO.21. The fusion protein PLIN1-Snf7 comprises a localization peptide of a lipid droplet and a Snf7 protein in an ESCRT-III complex. The localization peptide of the lipid droplet has the amino acid sequence shown in SEQID NO.19, and the Snf7 protein has the amino acid sequence shown in SEQ ID NO.20.

[0175] 1.1. Synthesis of target gene fragment encoding fusion protein PLIN1-Snf7

[0176] S1.11. Synthesis of the first gene fragment: human cDNA (NCBI accession number: NC_000015.10) was used as a template, and the template was amplified by PCR 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 product obtained by PCR amplification was purified and recovered by agarose gel electrophoresis to obtain the first gene fragment, which contained a tandem DH2 promoter homologous sequence and a nucleotide sequence for encoding a localization peptide of liquid lipid droplets, and the nucleotide sequence of the first gene fragment was shown in SEQ ID NO.3;

[0177] S1.12. Synthesizing the second gene fragment: using the Saccharomyces cerevisiae genome as a template, using primer pair F2-R2 to perform PCR amplification on the template, wherein the nucleotide sequence of F2 is as shown in SEQ ID NO.4, and the nucleotide sequence of R2 is as shown in SEQ ID NO.5, and the product obtained by PCR amplification is purified and recovered by agarose gel electrophoresis to obtain the second gene fragment, which comprises a homologous sequence of a nucleotide sequence for encoding a localization peptide of a liquid lipid droplet, a nucleotide sequence for encoding a fusion junction, a nucleotide sequence encoding a Snf7 protein, and a CYC1 terminator homologous sequence, which are sequentially connected in series, and the nucleotide sequence of the second gene fragment is as shown in SEQ ID NO.6;

[0178] S1.13, synthesizing the third gene fragment: using the Saccharomyces cerevisiae genome as a template, using primer pair F3-R3 to perform PCR amplification on the template, wherein the nucleotide sequence of F3 is as shown in SEQ ID NO.7, and the nucleotide sequence of R3 is as shown in SEQ ID NO.8, and the product obtained by PCR amplification is purified and recovered by agarose gel electrophoresis to obtain the third gene fragment, the third gene fragment comprises a tandem integration site 511b upstream homologous sequence and ADH2 promoter, and the nucleotide sequence of the third gene fragment is as shown in SEQ ID NO.9;

[0179] S1.14, synthesis of the fourth gene fragment: using the Saccharomyces cerevisiae genome as a template, using primer pair F4-R4 to perform PCR amplification on the template, 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, and the product obtained by PCR amplification is purified and recovered by agarose gel electrophoresis to obtain the fourth gene fragment, the fourth gene fragment comprises a tandem CYC1 terminator and a downstream homologous sequence of the integration site 511b, and the nucleotide sequence of the fourth gene fragment is shown in SEQ ID NO.12;

[0180] S1.15. Synthesis of the fifth gene fragment: Using the Saccharomyces cerevisiae genome as a template, the template was amplified by PCR using primer pair F5-R5, wherein the nucleotide sequence of F5 is as shown in SEQ ID NO.13, and the nucleotide sequence of R5 is as shown in SEQ ID NO.14. The product obtained by PCR amplification was purified and recovered by agarose gel electrophoresis to obtain the fifth gene fragment, which includes the upstream homology arm sequence of the integration site 511b, and the nucleotide sequence of the fifth gene fragment is as shown in SEQ ID NO.15;

[0181] S1.16. Synthesis of the sixth gene fragment: Using the Saccharomyces cerevisiae genome as a template, the template was amplified by PCR 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 product obtained by PCR amplification was purified and recovered by agarose gel electrophoresis to obtain the sixth gene fragment, which includes the downstream homology arm sequence of the integration site 511b, and the nucleotide sequence of the sixth gene fragment is shown in SEQ ID NO.18;

[0182] S1.17, using primer F5 and primer R6 as a primer pair, the first gene fragment to the sixth gene fragment were subjected to fusion PCR to obtain the target gene fragment, such as Figure 2 As shown, the target gene fragment includes 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).

[0183] 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:

[0184] Table 3 PCR reaction system list

[0185] Reagent name Dosage / μL 2×Phanta Max Master Mix 25 Forward primer (10 μM) 2 Reverse primer (10 μM) 2 template 1 Double distilled water (ddH2O) 20 Overall system 50

[0186] Compared with the PCR reaction system in Table 1, the difference between the reaction system of 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.

[0187] In step S1.11 to step S1.16, the reaction procedure of PCR is: ① pre-denaturation at 95℃ for 3min; ② denaturation at 95℃ for 15s; ③ annealing at 55℃ for 15s; ④ extension at 72℃ for 1min; ⑤ repeat ② to ④ for 35 cycles; ⑥ 72℃ for 5min.

[0188] 1.2. Construction of recombinant Sc-1

[0189] S1.21, preparation of Saccharomyces cerevisiae competent medium: pick Saccharomyces cerevisiae and inoculate it into 5 mL of YPD liquid medium, then place it in the shaking culture at 30℃ and 220r / min for 12h, then transfer the activated Saccharomyces cerevisiae to 50 mL of YPD liquid medium and change 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 mixture 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, the mixture is mixed and 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;

[0190] S1.22, transform the target gene fragment into Saccharomyces cerevisiae competent medium: take 30 μL of Saccharomyces cerevisiae competent medium prepared in step S1.21 into a 1.5 mL centrifuge tube, add 240 μL of PEG3350 aqueous solution (50 g of PEG3350 is added to pure water to make up to 100 mL), 36 μL of 1 mol / L lithium acetate, 5 μL of 10 mg / mL single-stranded fish sperm DNA (before use, it needs to be boiled at 99°C for 5-10 min and then immediately placed on ice), 1 μg of the target gene fragment synthesized in step S1.17, 300 μL of PEG3350 aqueous solution (50 g of PEG3350 is added to pure water to make up to 100 mL), 36 μL of 1 mol / L lithium acetate, 5 μL of 10 mg / mL single-stranded fish sperm DNA (before use, it needs to be boiled at 99°C for 5-10 min and then immediately placed on ice), 1 μg of the target gene fragment synthesized in step S1.17, ng of pCUT (511b-ura) plasmid and 70 μL of pure water, mix well to obtain the transformation system; first culture the transformation system at 30°C for 30 min, and then heat shock at 42°C for 40 min; centrifuge the transformation solution after heat shock at 5000 r / min for 1 min, discard the supernatant, add 1 mL of water and mix well, then centrifuge at 5000 r / min for 1 min, discard the supernatant again, add 100 μL of pure water and mix well to obtain the bacterial solution; evenly spread the bacterial solution on the SC-URA culture medium plate, and culture at 30°C for 48 h;

[0191] S1.23, verification: pick a single clone on the transformation plate and culture it in SC-URA liquid culture medium for 12h, then take 200μL of bacterial solution and centrifuge it at 5000r / min for 1min, collect the supernatant, add 100μL of 20mmol / L NaOH aqueous solution to the supernatant, place it at 99℃ for lysis for 20min, and then immediately place it on ice for 5min to obtain bacterial lysate; prepare a verification PCR system, the verification PCR system is shown in Table 4 below, and the reaction procedure of the verification PCR is: ① pre-denaturation at 94℃ for 2min; ② denaturation at 98℃ for 10s; ③ annealing at 55℃ for 30s; ④ extension at 72℃ for 2min; ⑤ repeat ②~④ for 35 cycles; ⑥ 72℃, 5min; use 1% agarose nucleic acid electrophoresis for the PCR product, the target band size is 2339bp, and the PCR product with the correct fragment size is subjected to Sanger first-generation sequencing. The clone with the correct sequencing result is the recombinant Sc-1.

[0192] Table 4 Verification PCR reaction system list

[0193]

[0194]

[0195] Among them, 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.

[0196] Example 2

[0197] This embodiment provides a recombinant Sc-2 and a construction method thereof. The recombinant Sc-2 can express a fusion protein PLIN1-STP22. The fusion protein PLIN1-STP22 comprises a localization peptide of a lipid droplet and a STP22 protein in an ESCRT-Ⅰ complex. The localization peptide of the lipid droplet 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.

[0198] 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 comprises 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 joint, 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.

[0199] Example 3

[0200] This embodiment provides a recombinant Sc-3 and a construction method thereof. The recombinant Sc-3 can express a fusion protein PLIN1-VPS25. The fusion protein PLIN1-VPS25 comprises a localization peptide of a lipid droplet and a VPS25 protein in an ESCRT-Ⅱ complex. The localization peptide of the lipid droplet 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.

[0201] 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 comprises 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 joint, the nucleotide sequence encoding the VPS25 protein, the CYC1 terminator and the upstream homology arm sequence (511b-HoR) of the integration site 511b, which are connected in series.

[0202] Example 4

[0203] This embodiment provides a recombinant Sc-4 and a construction method thereof. The recombinant Sc-4 can express the fusion protein PLIN1-VTA1. The fusion protein PLIN1-VTA1 comprises a localization peptide of a lipid droplet and a VTA1 protein in a Vps4-VTA1 complex. The localization peptide of the lipid droplet 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.

[0204] 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 by "nucleotide sequence encoding VTA1 protein", and the target gene fragment in step S1.17 is replaced by the nucleotide sequence shown in SEQ ID NO.29, and the target gene fragment comprises 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 joint, 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.

[0205] Comparative Example 1

[0206] This comparative example provides a recombinant Sc-5 and a construction method thereof. The recombinant Sc-5 can overexpress a localization peptide of a lipid droplet of PLIN1, and the localization peptide of the lipid droplet has an amino acid sequence shown in SEQ ID NO.19.

[0207] 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 an upstream homologous arm sequence (511b-HoL) of the integration site 511b, an ADH2 promoter, a nucleotide sequence for encoding a localization peptide of liquid lipid droplets, a CYC1 terminator and an upstream homologous arm sequence (511b-HoR) of the integration site 511b connected in series".

[0208] Comparative Example 2

[0209] 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, and the Snf7 protein has an amino acid sequence shown in SEQ ID NO.20.

[0210] Compared with the construction method of the recombinant Sc-1 in Example 1, the difference in the construction method of the 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 the Snf7 protein, the CYC1 terminator and the upstream homologous arm sequence (511b-HoR) of the integration site 511b connected in series".

[0211] Experimental example

[0212] The recombinants of Examples 1 to 4, Comparative Examples 1 to 4, and Saccharomyces cerevisiae YB / I / E extra I (as a control) were fermented and cultured under the same conditions to produce β-carotene, and the differences in the extracellular yields of β-carotene produced by the fermentation of the recombinants were compared.

[0213] The fermentation culture of each recombinant comprises the following steps:

[0214] S101. Inoculate the recombinant into 5 mL of YPD liquid medium and culture at 30°C and 220 r / min until OD 600 The value of is 0.5, and the first culture solution is obtained;

[0215] 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 r / min for 6 days to obtain a second culture solution;

[0216] S103, centrifuging the second culture solution at a speed of 4000 r / min for 10 min, collecting the supernatant, which contains β-carotene;

[0217] S104, using HPLC technology to detect the content of β-carotene in the supernatant obtained in step S103.

[0218] The extracellular yield of β-carotene produced by each recombinant fermentation is shown in Table 1 below:

[0219] Table 1 List of extracellular yields of β-carotene produced by various recombinant fermentations

[0220]

[0221] As shown in Table 1, compared with the extracellular yield of β-carotene produced by the fermentation of the recombinants in Comparative Examples 1 and 2 and Saccharomyces cerevisiae YB / I / E extra I, the extracellular yield of β-carotene produced by the fermentation of 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, Figure 3 and Figure 4 It can be seen that the extracellular production of β-carotene by the Sc-1 recombinant fermentation is 9 times the extracellular production of β-carotene by the fermentation of Saccharomyces cerevisiae YB / I / E extra I. It can be seen that for the recombinant that can express and produce the fusion protein (including the localization peptide of the lipid droplet and the ESCRT protein), the fusion protein can be localized on the surface of the lipid droplets in the recombinant, so that the lipid droplets storing β-carotene enter the sorting link through the ESCRT pathway, and finally enter the exosomes, and are secreted to the extracellular space through the exosomes, effectively increasing the extracellular production of β-carotene.

[0222] 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.

[0223] The above is a detailed introduction to a fusion protein provided by the present invention 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 embodiments is only used to help understand the technical solutions and core ideas of the present application; ordinary technicians in this field should understand that they can still modify the technical solutions recorded in the aforementioned embodiments, or replace some of the technical features therein by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A fusion protein, characterized in that 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, characterized in that 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; 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 The one or more proteins in the Vps4-Vta1 complex are selected from one or more of Vta1, Vps4, SKD1, SKD2 and LIP5; and / or 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 or 2, characterized in that The first subcellular structure is selected from one or more of lipid droplets, peroxisomes, lysosomes, mitochondria, centrosomes, Golgi bodies and microsomes; and / or 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 respectively.

4. The fusion protein according to claim 3, characterized in that 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 substituting at least one amino acid, and / or deleting at least one amino acid, and / or adding at least one amino acid from the amino acid sequence shown in SEQ ID NO.19, and has the function of localizing on the surface of the lipid droplet; And / or, 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 to 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; And / or, 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.

5. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleotide sequence encoding a fusion protein as described in any one of claims 1 to 4; Optionally, the nucleic acid molecule further comprises one or more of a promoter, an intron, an exon, a terminator and an enhancer; and / or, 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.

6. A recombinant expression vector, characterized in that: comprising a vector and the nucleic acid molecule according to claim 5, wherein the nucleic acid molecule is loaded on the vector; 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.

7. A recombinant, characterized in that The recombinant comprises a host, and the nucleic acid molecule according to claim 5 or the recombinant expression vector according to claim 6 introduced into the body of the host; Optionally, the host is yeast.

8. A method for preparing a fusion protein, characterized in that: The method comprises the steps of: fermenting and culturing the recombinant according to claim 7 to express and obtain the fusion protein.

9. The preparation method according to claim 8, characterized in that: The method for introducing a nucleic acid molecule into the body of the host as claimed in claim 5 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 as described in any one of claims 1 to 4, 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.

10. A method for preparing a metabolite, characterized in that: The method comprises the steps of: fermenting and culturing the recombinant as claimed in claim 7 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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