Patent Application
20240002888
The present disclosure relates to a recombinant microorganism including a polynucleotide encoding a target product-binding protein fused to a secretion signal sequence, a composition including the recombinant microorganism, and a method of producing a target product by using the recombinant microorganism.
Many products are produced by using recombinant microorganisms. For example, by using recombinant E. coli or yeast cells including S. cerevisiae, several products are produced.
However, some products are present in cells after being biosynthesized in recombinant microorganisms. Such products present in cells require an additional step of disrupting the cells to be isolated therefrom.
Therefore, there is a need for a recombinant microorganism capable of secreting a product present in a cell after being biosynthesized to the outside of the cell and for a method of producing a product by using the recombinant microorganism.
One aspect provides a recombinant microorganism comprising a polynucleotide encoding a target product-binding protein fused to a secretion signal sequence.
Another aspect provides a composition for use in producing a target product, comprising a carrier and the recombinant microorganism comprising a polynucleotide encoding a target product-binding protein fused to a secretion signal sequence.
Another aspect provides a method of producing a target product, the method comprising culturing the recombinant microorganism in a medium, wherein the recombinant microorganism comprises a polynucleotide encoding a target product-binding protein fused to a secretion signal sequence.
One aspect provides a recombinant microorganism comprising a polynucleotide encoding a target product-binding protein fused to a secretion signal sequence.
The polynucleotide may be present in the genome or outside of the genome of the recombinant microorganism. The polynucleotide may be operably linked to a regulatory sequence. The regulatory sequence may be a sequence necessary for biosynthesis or production of the protein from the polynucleotide. The regulatory sequence may be a transcriptional regulatory sequence, a translational regulatory sequence, and a post-translational modification regulatory sequence. The regulatory sequence may be a promoter, an operator, a terminator, an enhancer, a ribosome-binding site, or a combination thereof. The regulatory sequence may be endogenously present before the recombinant microorganism is recombined, or may be an exogenous regulatory sequence. The regulatory sequence may be a regulatory sequence of a gene encoding a naked target product-binding protein to which the secretion signal sequence is not fused, or may be a regulatory sequence of a gene encoding other proteins. The polynucleotide may be included in multiple copies in the cell. The polynucleotide may be, for example, included in copies in a range of 2 or more, 5 or more, 10 or more, 50 or more, or 100 or more copies or in a range of 2 to 1,000, 2 to 500, 2 to 100, 2 to 50, 2 to 10, 2 to 5, 5 to 100, 5 to 50, 5 to 10, 5 to 100, 5 to 10, 10 to 100, or 10 to 50 in the cell. The polynucleotide may be linked to the regulatory sequence to enable expression. The polynucleotide may be linked to the regulatory sequence that allows expression at an increased level compared to an expression level of the endogenous polynucleotide. For example, the polynucleotide may be linked to a promoter having stronger ability for initiation of transcription than an endogenous promoter.
The polynucleotide may also include a nucleotide sequence encoding a tag sequence for use in isolating the complex or the dissociated target product-binding protein secreted extracellularly. The tag sequence may be an oligopeptide. The tag sequence may include a sequence of two or more amino acids, for example, 2 to 50, 2 to 30, 2 to 20, 2 to 15, or 2 to 10 amino acids. The tag sequence may be, for example, a His tag sequence. The His tag may be linked to the N-terminus or the C-terminus of the target product-binding protein. The tag may be 6×His.
The polynucleotide may encode an amino acid sequence in which a secretion signal sequence is fused to the N-terminus of the target product-binding protein.
The polynucleotide may be introduced into a microbial cell before being recombined by a genetic modification method known in the art. The genetic modification may include transformation, transduction, transfection, or electroporation. In addition, the polynucleotide may be produced by gene editing of a gene in a microbial cell before being recombined. The gene editing may be performed by a known method in the art, such as clustered regularly interspaced short palindromic repeats (CRISPR) gene editing.
The recombinant microorganism may have an ability to secrete the target product-binding protein fused to the secretion signal sequence. The recombinant microorganism may have an ability to produce the target product. The recombinant microorganism may have an increased ability to produce the target product. The recombinant microorganism may be secreted extracellularly in the form of a complex in which the target product and the target product-binding protein are bound. Formation of the complex may be performed intracellularly. The formation of the complex may be achieved by cells in a cellular metabolic process without manipulation from the outside. In the complex, the bond between the target product and the target product-binding protein may be a non-covalent bond. The non-covalent bond may include a hydrophobic bond, a hydrogen bond, or an ionic bond. The complex may be dissociated in a liquid medium under conditions suitable for dissociating the bond to form a dissociated target product and the target product-binding protein, respectively. A target product-binding protein that is not fused to the secretion signal sequence may be present intracellularly, for example, in the cytoplasm of a natural host cell, and thus may not be secreted extracellularly. The target product-binding protein not fused to the secretion signal sequence may not be linked to the secretion signal sequence in the natural host cell.
The target product may be a metabolite. The metabolite may be an endogenous metabolite or a metabolite synthesized by a synthesis pathway thereof introduced by genetic modification. The metabolite may be a lipid, a polysaccharide, a protein, or a small molecule compound. The target product may be a lipid. The lipid may be a terpenoid. The term “terpenoid” as used herein refers to a compound synthesized by using isoprene as a unit. The number of carbon atoms in the terpenoid may be in a range of 5 to 100, 10 to 100, 15 to 100, 30 to 100, 5 to 60, 10 to 60, 15 to 60, 30 to 60, 5 to 45, 10 to 45, 15 to 45 carbon atoms, 30 to 60, 5 to 30, 10 to 30, 15 to 30, or 5 to 45. The terpenoid may include oxygen molecules. In addition, the terpenoid may be a hydrocarbon that does not contain oxygen molecules. The terpenoid may be monoterpene, diterpene, sesquiterpene, triterpene, or tetraterpene. The terpenoid may be a cyclic compound or a non-cyclic compound. The terpenoid may be selected from the following 43 compounds, i.e., 2,3-oxidosqualene, myrcene, (z)-ocimene, linalool, geraniol, nerol, limonene, menthol, α-pinene, camphor, carvacrol, carvone, β-farnesene, nerolidol, β-bisabolene, valencene, amorphadiene, capsidiol, α-cedrane, artemisinin, phytane, phytol, camphorene, retinol, labdane, clerodane, pimarane, abietane, tigliane, kaurane, taxane, squalene, lanosterol, α-amyrin, β-amyrin, hopane, oleanolic acid, lycopene, rubixanthin, β-carotene, lutein, zeaxanthin, and astaxanthin. The target product may be squalene or beta-carotene. The recombinant microorganism may have an enhanced biosynthetic pathway of terpenoid. The recombinant microorganism may have an enhanced biosynthetic pathway of one or more of the 43 compounds.
The target product-binding protein may bind to the metabolite. The target product-binding protein may specifically bind to the metabolite. The target product-binding protein may be a lipid-binding protein. The target product-binding protein may be a cytoplasmic lipid-binding protein. The lipid-binding protein may be selected from the group consisting of a supernatant protein factor (SPF), a phosphatidylinositol transfer protein (Sec14), an alpha-tocopherol transfer protein (TTP), a cellular retinal binding protein, a squid retinal binding protein, and fragments thereof having target product-binding ability. The lipid-binding protein may be derived from any cell. For example, the lipid-binding protein may be derived from a mammalian cell. The SPF and the TTP may be derived from a human or a mouse. The human-derived SPF has a sequence of 403 amino acids, and may have an N-terminal domain with lipid binding ability and a C-terminal domain with a jelly-roll motif, i.e., a golgi dynamics domain (GOLD). The SPF may be a truncated SPF (tSPF) having squalene binding ability and a C-terminal domain in which the amino acid sequences at positions 276 to 403 have been removed by cleavage. That is, the tSPF may consist of the sequence of amino acids 1 to 275 of the SPF. The SPF and the tSPF may have amino acid sequences of SEQ ID NOs: 1 and 2, respectively. Nucleotides encoding the SPF and the tSPF may have nucleotide sequences of SEQ ID NOs: 3 and 4, respectively.
The secretion signal sequence represents an amino acid sequence that secretes a protein expressed in a cell to the outside of the cell. The secretion signal sequence may be a secretion signal sequence derived from a eukaryotic cell or a prokaryotic cell. The secretion signal sequence may be any amino acid sequence that can secrete the target product-binding protein from the recombinant microorganism to the outside of the cell. The secretion signal sequence may be derived from the same or different cell from the recombinant microbial cell. The secretion signal sequence may be a secretion signal sequence derived from animals, plants, yeast, or bacteria. The secretion signal sequence may be one or more selected from the group consisting of Suc2, Pho5, MFα, Inut and Mel1 secretion signal sequences. The Suc2, Pho5, and MFα secretion signal sequences may have amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively. Nucleotides encoding the Suc2, Pho5, and MFα secretion signal sequences may have nucleotide sequences of SEQ ID NOs: 8, 9, and 10, respectively.
The recombinant microorganism may be a eukaryotic cell or a prokaryotic cell. The eukaryotic cell may be an animal cell, a plant cell, or a fungal cell. The fungus may be yeast. The yeast cell may belong to the genus Saccharomyces. A cell of the genus Saccharomyces may be Saccharomyces cerevisiae. The prokaryotic cell may be a bacterium. The bacteria may belong to the genus Escherichia, the genus Corynebacterium, or the genus Bacillus. In addition, the bacteria may be lactic acid bacteria belonging to, for example, the genus Lactobacillus or the genus Lactococcus. The bacteria may be Escherichia coli, Corynebacterium glutamicum, or Bacillus subtilus.
The recombinant microorganism may be genetically modified to increase the expression of the polynucleotide encoding the target product, or may have an enhanced metabolic pathway for producing the target product. The recombinant microorganism may have an increased ability to produce the target product compared to cells before transformation. The genetic modification may include an increase in the number of copies of the polynucleotide encoding the target product. The genetic modification may include modification of the regulatory sequence to increase the expression of the polynucleotide encoding the target product. In addition, the recombinant microorganism may have an enhanced metabolic pathway for producing the target product. The metabolic pathway for producing the target product may be enhanced by genetic modification that increases the expression of the polynucleotide encoding a factor, such as a protein or an enzyme, involved in the biosynthesis of the target product. The genetic modification may include an increase in the number of copies of the polynucleotide encoding a factor, such as a protein or an enzyme, involved in the biosynthesis of the target product, or modification of the regulatory sequence of the polynucleotide.
The recombinant microorganism may also have an attenuated metabolic pathway that inhibits the production of the target product. The metabolic pathway may be, for example, a pathway that degrades the target product or a feedback inhibition pathway.
In an embodiment, the target product-binding protein may be SPF or a ligand binding fragment thereof. The target product-binding protein may be a polypeptide including amino acids, such as L84, I103, L106, A108, L111, L112, L120, L121, K124, I151, Y153, C155, L158, H162, A167, V168, A170, Y171, F174, L175, L186, L189, F198, A201, Y202, 1205, L209, T213, and 1217 in the amino acid sequence of the SPF of SEQ ID NO: 1. The recombinant microorganism may belong to the genus Saccharomyces, and for example, may be Saccharomyces cerevisiae. The recombinant microorganism may have an enhanced biosynthesis pathway of mevalonic acid or beta-carotene. The recombinant microorganism may include a genetic modification that increases activity of one or more of HMG-CoA and ERG20. The recombinant microorganism may include a genetic modification that increases expression of a gene encoding HMG-CoA or ERG20. The genetic modification may include an increase in the number of copies of the gene. In the recombinant microorganism, one or more of trp1, leu2, his3, and ypl062w genes may be inactivated. The genetic modification may include not only completely eliminating but also reducing the expression of the gene to be inactivated. The inactivation may be deletion of one or more of the trp1, leu2, his3 and ypl062w genes. HMG-CoA, ERG20, trp1, leu2, his3, and ypl062w may have amino acid sequences of SEQ ID NOs: 11, 12, 13, 14, 15, and 16, respectively. HMG-CoA, ERG20, trp1 gene, leu2 gene, his3 gene, and ypl062w gene may have nucleotide sequences of SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively.
Another aspect provides a composition for use in producing a target product, the composition comprising a carrier and the recombinant microorganism comprising a polynucleotide encoding a target product-binding protein fused to a secretion signal sequence.
The recombinant microorganism comprising a polynucleotide encoding a target product-binding protein fused to a secretion signal sequence is defined the same as described above. The carrier may be a medium used for culturing the microorganism, a buffer, or a storage solution.
Another aspect provides a method of producing a target product, the method comprising culturing the recombinant microorganism in a medium, wherein the recombinant microorganism comprises a polynucleotide encoding a target product-binding protein fused to a secretion signal sequence.
The recombinant microorganism comprising a polynucleotide encoding a target product-binding protein fused to a secretion signal sequence is defined the same as described above.
The culturing may include incubating a mixture including the recombinant microorganism and a medium under conditions suitable for growing the recombinant microorganism and producing a target product-binding protein fused to the secretory signal sequence.
The culturing may be performed in a medium containing a carbon source, for example, glucose. The medium used for culturing the microorganisms may be any conventional medium suitable for the growth of the selected recombinant cells, such as a minimal or complex medium containing appropriate supplements. Such a suitable medium may be available from commercial sellers, or can be prepared according to known methods.
The medium may be a medium that can satisfy the requirements of specific microorganisms according to the selected target product. The medium may include a component selected from the group consisting of a carbon source, a nitrogen source, a salt, a trace element, and a combination thereof.
Conditions of the culture may be appropriately adjusted to suit the production of a selected target product, for example, terpenoid such as squalene. The culturing may be performed under aerobic conditions for cell proliferation. The culturing may be performed without stirring or with stirring. The culturing may be performed in a batch or a fed batch, or continuously. The continuous culturing may refer to continuous culturing while not only removing some of the microorganism or supernatant from the culture, but also supplementing a medium. The recombinant microorganism may secrete the target product extracellularly in the form of a complex with the target product-binding protein, and that is, may be separated from the supernatant from which the cells are removed from the culture.
The culturing may refer to culturing in a medium containing a compound having high solubility of a target product. The solubility may be higher than that for the medium or water. The solubility for target product may be greater than 1.2, 1.5, 2.0, 3.0, 5.0, 7.5, or 10 times higher than the solubility for the medium or water, or may be 1.2 to 10, 1.5 to 10, 2.0 to 10, 3.0 to 10, 5.0 to 10, or 7.5 to 10 times higher than the solubility for the medium or water. The target product may be a lipid, such as terpenoid, and the compound having high solubility of the target product may be an oily compound having high solubility of the lipid, such as terpenoid. The target product may be secreted extracellularly in the form of a target product-target product-binding protein complex during culturing, and in the extracellular medium, the complex may exist in a state in which the complex is dissociated into the target product and the target product-binding protein and in a dynamic equilibrium state. Accordingly, a part of the target product dissociated in the dynamic equilibrium state may be distributed to a compound layer having high solubility of the target product. For example, when the target product is a lipid, such as terpenoid, and the culturing is performed in a medium containing an oily compound, the target product may be distributed to the oily compound in the medium after being secreted extracellularly. The oily compound may be an alkane solvent, for example, a C1-C60 alkane, a C6-C60 alkane, or a C3-C30 alkane. The solvent may be hexane, heptane, decane, dodecane, or hexadecane. In addition, the oily compound may be vegetable oil. The vegetable oil may be olive oil, canola oil, or essential oil. The oily compound may be a compound that is easy to separate from the target product due to a large boiling point difference from the target product.
The term “conditions for culture” as used herein refers to conditions for culturing microorganisms. Such conditions for culture may include, for example, carbon source, nitrogen source, or oxygen conditions used by microorganisms. A carbon source available to yeast may include monosaccharides, disaccharides, or polysaccharides. The carbon source is an assimilatable sugar, and may include glucose, fructose, mannose, or galactose. The nitrogen source may include an organic nitrogen compound or an inorganic nitrogen compound. The nitrogen source may include an amino acid, an amide, an amine, a nitrate, or an ammonium salt. Oxygen conditions for culturing microorganisms may include aerobic conditions with normal oxygen partial pressure, hypoxic conditions containing 0.1% to 10% oxygen in the atmosphere, or anaerobic conditions without oxygen.
The method may include isolating the target product. The isolating of the target product may not include cell lysis. The isolating of the target product may include separating a supernatant from a culture. The separating may include separating the target product from the supernatant. The isolating of the target product may include separating the target product from the complex of the target product and the target product-binding protein. The separating of the target product from the complex of the target product and the target product-binding protein may include incubating the culture or supernatant containing the complex in a liquid medium under conditions suitable for dissociating the complex into the target product and the target product-binding protein. The conditions may be appropriately selected depending on the selected target product and target product-binding protein. For example, the target product may be a terpenoid compound, such as squalene or beta-carotene, and the isolating of the target product may include incubating the complex in a hydrophobic solvent, for example, with an oily compound. The solvent may be an alkane, such as a C1-C60 alkane, a C6-C60 alkane, or a C3-C30 alkane. The solvent may be hexane, heptane, decane, dodecane, or hexadecane. The isolating of the target product may be performed simultaneously with the culturing of the recombinant microorganism.
In an embodiment, the target product may be a compound selected from the group consisting of 2,3-oxidosqualene, myrcene, (z)-ocimene, linalool, geraniol, nerol, limonene, menthol, α-pinene, camphor, carvacrol, carvone, β-farnesene, nerolidol, β-bisabolene, valencene, amorphadiene, capsidiol, α-cedrane, artemisinin, phytane, phytol, camphorene, retinol, labdane, clerodane, pimarane, abietane, tigliane, kaurane, taxane, squalene, lanosterol, α-amyrin, β-amyrin, hopane, oleanolic acid, lycopene, rubixanthin, β-carotene, lutein, zeaxanthin, and astaxanthin. The target product-binding protein may be selected from the group consisting of SPF, Sec14, TTP, an alpha-tocopherol transfer protein, a cellular retinal binding protein, a squid retinal binding protein, and fragments thereof having target product-binding ability. The recombinant microorganism may be a yeast cell, for example, of the genus Saccharomyces. The microorganism belonging to the genus Saccharomyces may be S. cerevisiae.
A recombinant microorganism according to one aspect can be used to efficiently produce a target product.
A composition for use in producing a target product according to one aspect can be used to efficiently produce a target product.
According to a method of producing a target product according to one aspect, a target product can be efficiently produced.
Hereinafter, the present disclosure will be described in detail with reference to Examples below. However, these Examples are for illustrative purposes only, and the scope of the present disclosure is not intended to be limited by these Examples.
In this example, recombinant yeast cells were prepared by introducing a target product-binding protein fused to a secretion signal sequence into yeast cells, and a target product was produced by using the prepared recombinant yeast cells, and the secretion ability of the target product was confirmed. Saccharomyces cerevisiae was used as the yeast cell, and Suc2, Pho5, and MFα secretion signal sequences were used as secretion signal sequences, respectively. In addition, squalene was used as the target product, and tSPF, which is a truncated fragment of supernatant protein factor (SPF), was used as the target product-binding protein. tSPF is a squalene-binding protein corresponding to the N-terminal domain of SPF with lipid-binding ability. tSPF corresponds to amino acid sequences 1 to 275 in SPF having the amino acid sequence of SEQ ID NO: 1.
(1.1) Preparation of Yeast Cells with Enhanced Biosynthetic Pathway of Squalene
Yeast cells were S. cerevisiae, and the wild-type strain was CEN.PK2-1D [(MATα ura3-52; trp1-289; leu2-3,112; his3Δ 1; MAL2-8; SUC2) (EUROSCARF Accession Number: 30000B). In the strain, genes encoding phosphoribosylanthranilate isomerase (trp1), 3-isopropylmalate dehydrogenase (leu2), and imidazoleglycerol-phosphate dehydratase (his3) were inactivated. trp1, leu2, and his3 may have amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively. The genes encoding trp1, leu2, and his3 may have nucleotide sequences of SEQ ID NOs: 19, 20, and 21, respectively. Squalene is a terpenoid that is synthesized in yeast cells through a mevalonic acid biosynthetic pathway. To increase the squalene production ability, the following genetic modifications were introduced.
First, to the sites where the genes, i.e., trp1, leu2, and his3, were deleted from the strain, the tHMG1 gene encoding S. cerevisiae-derived HMG-CoA reductase (β-hydroxy β-methylglutaryl-CoA reductase) and a gene encoding S. cerevisiae-derived farnesyl pyrophosphate synthetase (ERG20) were introduced. The tHMG1 and ERG20 may have amino acid sequences of SEQ ID NOs: 11 and 12, respectively. The genes encoding tHMG1 and ERG20 may have nucleotide sequences of SEQ ID NOs: 17 and 18, respectively. The HMG-CoA reductase is an enzyme that catalyzes the reaction of converting HMG-CoA to mevalonate. The ERG20 is an enzyme that catalyzes the conversion of GPP to farnesyl pyrophosphate (FPP). Both HMG-CoA and FPP may be precursors for the synthesis of squalene.
In addition, in the genome of the strain, the tHMG1 gene was introduced to the site of ypl062w while simultaneously deleting a gene encoding ypl062w. Due to the deletion of the ypl062w gene, the level of acetyl-CoA, which is an early substrate in the biosynthetic pathway of mevaloate, may increase. The ypl062w may have the amino acid sequence of SEQ ID NO: 16. The gene encoding ypl062w may have the nucleotide sequence of SEQ ID NO: 22.
As a result, a recombinant S. cerevisiae strain, named SQ, was obtained. A more detailed process for producing the SQ strain is disclosed in Korean patent KR 10-2176555, and the contents thereof are incorporated herein by reference.
(1.2) Preparation of Yeast Cells to which Gene Encoding Squalene-Binding Protein Fused to Secretion Signal Sequence has been Introduced
Three different signal peptides were used for the secretion signal sequence, and tSPF was used for the squalene-binding protein. Specifically, a polynucleotide encoding a fusion protein was obtained, in which three different signal peptides, i.e., Suc2, Pho5, and MFα signal peptides, were each fused to the N-terminus of tSPF. The fusion protein may include a 6×His tag sequence at the C-terminus.
The Suc2, Pho5, and MFα signal peptides may have the amino acid sequences of SEQ ID NOs: 5, 6, and 7, respectively. Nucleotides encoding the Suc2, Pho5, and MFα signal peptides may have nucleotide sequences of SEQ ID NOs: 8, 9, and 10, respectively. The Suc2, Pho5, and MFα signal peptides may be amino acid sequences with the function of N-terminal signal peptides, and may secrete sucrose transporter protein (Suc2), an acid phosphatase (Pho5), and a yeast α-mating factor (MFα), respectively, into the outside the cell. The tSPF may have the amino acid sequence of SEQ ID NO: 2. The tSPF-encoding gene may have the nucleotide sequence of SEQ ID NO: 4. As a result, the obtained fusion proteins, i.e., Suc2-tSPF, Pho5-tSPF, and MFα-tSPF fusion proteins, may have amino acid sequences of SEQ ID NOs: 23, 24, and 25, respectively. Nucleotides encoding the Suc2-tSPF, Pho5-tSPF, and MFα-tSPF fusion proteins may have nucleotide sequences of SEQ ID NOs: 26, 27, and 28, respectively.
Next, the polynucleotide was introduced into the SpeI/XhoI restriction enzyme site of the p426-TEF1 (SEQ ID NO: 29) vector to obtain expression vectors pSEC-Suc2-tSPF, pSEC-Pho5-tSPF and pSEC-MFα-tSPF. As controls, the pSEC-tSPF vector to which the tSPF gene without the secretory signal sequence was introduced, and the empty p426-TEF1 vector were used. p426-TEF1 is a high copy number plasmid including a TEF1 promoter. The fusion protein is operably linked by the TEF1 promoter, and can be transcribed thereby.
The expression vectors were each introduced into the SQ strain by a heat shock transformation method to obtain SQ strains each transformed with three fusion protein genes. The expression vectors were present outside, independently from the genome of the SQ strain.
(1.3) Confirmation of Squalene Production Ability and Squalene Secretion Ability by Recombinant Yeast Cells
The SQ strains transformed with the pSEC-Suc2-tSPF, pSEC-Pho5-tSPF, and pSEC-MFα-tSPF expression vectors were cultured in flask (250 L) containing 50 mL of minimal medium (e.g., YSC medium; 0.67% (w/v) yeast nitrogen base without amino acids (BD Difco, USA), 0.19% (w/v) yeast synthetic drop-out medium supplements without uracil (Sigma-Aldrich, USA)) while stirring at 250 rpm at 30° C.
After culturing for 72 hours and 144 hours, the culture was centrifuged at 4,000 rpm to be separated into cells, a culture supernatant, and a dodecane layer. Then, the concentration of squalene in the separated cells and dodecane layer was measured. To measure the squalene concentration in the cells, the cells were resuspended in 600 μL of a mixed solution containing methanol and acetone at 1:1, transferred to a tube containing lysing matrix C (glass bead, MP Biomedicals, USA), and then underwent cell lysis by using a FastPrep-24 5G homogenizer (MP Biomedicals, USA). By centrifugation at 13,000 rpm, the lysed cells and intracellular metabolites including squalene dissolved from the lysed cells were separated.
Next, the squalene concentration in the cell lysate was measured. To measure the squalene concentration in the cell lysate, the concentration was measured by using an Agilent HPLC system after filtering the cell lysate through a 0.2 μm syringe filter.
In addition, the dodecane layer separated by centrifugation was also filtered in the same way, and then the squalene concentration was measured by using an HPLC system. Table 1 shows the squalene concentration in the cells or outside the cells, wherein the cells were produced by yeast cells including the polynucleotide encoding tSPF fused to the secretion signal sequence.
As shown in Table 1 and
The protein samples thus prepared, i.e. the cell lysates, were mixed with 5×SDS sample buffer and separated by 10% SDS-PAGE. The proteins separated on the SDS-PAGE gel were transferred to a PVDF membrane, and blocked by incubation for one day in TBST containing 4% skim milk. The PVDF membrane was reacted with a solution containing primary antibodies at room temperature for 1 hour, and then washed with a TBST solution three times. As the primary antibody, an anti-actin antibody or an anti-His tag antibody was used. The fusion protein included a 6×His tag sequence at the C-terminus. The washed PVDF membrane was reacted again in a solution containing peroxidase-conjugated anti-IgG antibodies at room temperature for 1 hour. The PVDF membrane in which the antibody-conjugated proteins were present was visualized by using the ChemiDoc imaging system.
The western blot results are shown in
(2.1) Preparation of Yeast Cells with Enhanced Biosynthetic Pathway of Beta-Carotene
A vector containing a beta-carotene biosynthesis gene was introduced into CEN.PK2-1D yeast strain described in Section 1.1 of Example 1 to prepare yeast cells with increased beta-carotene production ability.
In detail, the crtE, crtYB, crtI, and tHMG1 genes were amplified from the pMM494 vector (Addgene, Plasmid #100539) containing expression cassettes for the beta-carotene biosynthetic genes crtE, crtYB, crtI, and tHMG1, and these genes were introduced to the sites of restriction enzymes BamHI and SalI of p415-GPD vector (SEQ ID NO: 30), to construct p415-GPD vetor including the expression cassettes for crtE, crtYB, crtI, and tHMG1. The p415-GPD vector (ATCC 87358) is a low copy number expression vector including a GPD1 promoter. CrtE, crtYB, and crt proteins have the amino acid sequences of SEQ ID NOs: 31, 32, and 33, respectively. Polynucleotides encoding crtE, crtYB, and crt have nucleotide sequences of SEQ ID NOs: 34, 35, and 36, respectively. The crtE, crtYB, and crtI proteins derived from Xanthophyllomyces dendrohou, and have the following activities. CrtE is a gene encoding geranylgeranyl diphosphate that catalyzes a conversion reaction from farnesyl diphosphate to geranylgeranyl diphosphate. CrtYB includes crtB, which encodes phytoene synthase that catalyzes a conversion reaction from geranylgeranyl diphosphate to phytoene, and crtY, which encodes lycopene beta-cyclase that catalyzes a conversion from lycopene to beta-carotene. CrtI is a gene encoding phytoene desaturase that converts phytoene to lycopene.
(2.2) Preparation of Yeast Cells to which Gene Encoding Squalene-Binding Protein Fused to Secretion Signal Sequence has been Introduced
Through the same process as described in Section (1.2) of Example 1, the S. cerevisiae wild strain CEN.PK2-1D was co-transformed with the p415-BC vector manufactured in Section (1.2) and either pSEC-tSPF or pSEC-Suc2-tSPF, to manufacture yeast cells transformed with a vector expressing tSPF fused to a signal peptide.
(2.3) Confirmation of Beta-Carotene Production Ability and Beta-Carotene Secretion Ability by Recombinant Yeast Cells
CEN.PK2-1D yeast cells, which were co-transformed with the p415-BC vector obtained in Section (2.2) and either pSEC-tSPF or pSEC-Suc2-tSPF and were able to overproduce beta-carotene, were cultured for 144 hours under the same conditions as described in Section (1.3) of Example 1. Then, the amount of beta-carotene in the cultured cells and outside the cells was measured. The amount of beta-carotene was measured in the same method as the methods for cell lysis and dodecane layer separation used for the measurement of the squalene concentration. Table 2 and
As shown in Table 2 and
To identify the ligand-binding site of tSPF, the three-dimensional crystallographic structure of tSPF (PDB ID: 4OMK) was obtained from the RCSB Protein Data Bank. For the molecular docking analysis, the A-chain of 4ONK, from which squalene and water molecules bonded in the crystal structure were removed before simulation, was used. To construct a rigid protein structure from the crystallographic structure, one possible structure was intentionally selected for each amino acid. Next, the squalene-binding active site of the A-chain was determined by using the Computer Atlas of Surface Topology of protein (CASTp). Based on the amino acid sequence of SEQ ID NO: 1, the identified residues required for the squalene binding may be L84, I103, L106, A108, L111, L112, L120, L121, K124, I151, Y153, C155, L158, H162, A167, V168, A170, Y171, F174, L175, L186, L189, F198, A201, Y202, I205, L209, T213, and 1217. In this regard, the squalene-binding site was regarded to include these residues.
For the ligand selection, the following 43 terpenoids were selected from the PubChem Compound database in the sdf format: 2,3-oxidosqualene, myrcene, (z)-ocimene, linalool, geraniol, nerol, limonene, menthol, α-pinene, camphor, carvacrol, carvone, β-farnesene, nerolidol, β-bisabolene, valencene, amorphadiene, capsidiol, α-cedrane, artemisinin, phytane, phytol, camphorene, retinol, labdane, clerodane, pimarane, abietane, tigliane, kaurane, taxane, squalene, lanosterol, α-amyrin, β-amyrin, hopane, oleanolic acid, lycopene, rubixanthin, β-carotene, lutein, zeaxanthin, and astaxanthin.
In addition, glutamate and pyruvate were selected as control molecules. The downloaded sdf file was converted to a pdbqt format by Open Babel toolbox of the PyRx virtual tool. By using the information above, the molecular docking was performed with the AutoDock Vina to analyze the binding energy between tSPF and various terpenoid-based substances. The results are shown in Tables 3, 4, 5, and 6.
As shown in Tables 3, 4, 5, and 6, the difference in the binding energy between tSPF and each of the 43 terpenoid-based compounds was in a range of about −10.7 kcal/mol to about −6.1 kcal/mol, showing no significant difference even when compared with each of the binding energy of squalene of −10.5 kcal/mol and the binding energy of beta-carotene of −7.7 kcal/mol. These results indicate that the recombinant microorganism including the polynucleotide encoding tSPF fused to the above secretion signal sequence can be extensively applied to various terpenoid-based compounds.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0125985 | Sep 2020 | KR | national |
| 10-2021-0037445 | Mar 2021 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2021/008714 | 7/8/2021 | WO |
