Genetically modified cell producing ursodeoxycholic acid precursor and method for producing ursodeoxycholic acid precursors using same

Abstract

The present invention relates to genetically modified cells producing a ursodeoxycholic acid precursor and a method for producing a ursodeoxycholic acid precursor using same, and more specifically, provides a genetically modified cell and a method for producing an ursodeoxycholic acid (UDCA) precursor using same, wherein a biosynthetic pathway from cholesterol to cholic acid (CA) or chenodeoxycholic acid (CDCA), which are precursors of UDCA, is established in the cells, thereby producing intermediates in each step of the biosynthesis pathway, CA, or CDCA.

Description

Genetically modified cells producing ursodeoxycholic acid precursors and methods for producing ursodeoxycholic acid precursors using the same

[0001] The present invention relates to a genetically modified cell that produces an ursodeoxycholic acid (UDCA) precursor and a method for producing an ursodeoxycholic acid precursor using the same, and more particularly, to a genetically modified cell that constructs a biosynthetic pathway for cholic acid (CA) or chenodeoxycholic acid (CDCA), which are precursors of ursodeoxycholic acid, from cholesterol, and produces intermediate substances, CA or CDCA, at each step in the biosynthetic pathway, and a method for producing an UDCA precursor using the same.

[0002] Ursodeoxycholic acid (UDCA) is a tertiary bile acid that is synthesized in the liver, excreted in bile, metabolized by intestinal microorganisms, and then reabsorbed back into the liver. It is known to have effects such as liver detoxification, inhibition of hepatocyte damage, and antioxidant effects. According to a recent non-patent document 1, it has been reported that UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection.

[0003] As precursors of UDCA, its raw materials, cholic acid (CA) and chenodeoxycholic acid (CDCA), are extracted from animal-derived sources and chemically synthesized into UDCA. However, due to recent environmental issues that have increased supply and demand instability, there is a need for the development of technology that can stably produce raw materials using eco-friendly biotechnology.

[0004] Non-patent Document 2 discloses a method for synthesizing UDCA using hyodeoxycholic acid as a raw material through a nine-step reaction, yielding a total yield of 16%. While the raw materials used in this route are inexpensive and readily available, the steps are cumbersome and the yield is low. Furthermore, the use of metallic sodium during reduction makes the reaction more violent and potentially hazardous.

[0005] Against this backdrop, the inventors of the present invention have completed the present invention by introducing genetic resources of various origins into a yeast strain that is GRAS (Generally Recognized as Safe) to establish a biosynthetic pathway for a UDCA precursor, securing cells that stably produce UDCA precursors in an environmentally friendly manner, and developing a method for producing UDCA using the same.

[0006] [Prior Art Literature]

[0007] [Non-patent literature]

[0008] (Non-patent Document 1) Brevini, T., Maes, M., Webb, GJ et al. FXR inhibition may protect from SARS-CoV-2 infection by reducing ACE2. Nature 615, 134-142 (2023).

[0009] (Non-patent literature 2) Synthesis, 2016, 48: 588-594.

[0010] An object of the present invention is to provide a genetically modified cell producing a ursodeoxycholic acid precursor comprising at least one gene encoding an enzyme involved in a metabolic pathway for synthesizing a UDCA precursor from cholesterol, and a method for producing a UDCA precursor using the same.

[0011] Another object of the present invention is to provide a recombinant vector comprising at least one gene encoding an enzyme involved in a metabolic pathway for synthesizing a UDCA precursor from cholesterol.

[0012] In order to solve the above-described problem, the present invention provides a genetically modified cell producing an ursodeoxycholic acid precursor, which comprises at least one gene encoding an enzyme involved in a metabolic pathway for synthesizing an ursodeoxycholic acid (UDCA) precursor from cholesterol, and a recombinant vector comprising at least one gene encoding an enzyme involved in a metabolic pathway for synthesizing an ursodeoxycholic acid (UDCA) precursor from cholesterol, wherein the enzyme may be any one or more of the following i) to viii):

[0013] i) An enzyme that converts cholesterol into 7α-hydroxycholesterol;

[0014] ii) An enzyme that converts 7α-hydroxycholesterol to 7α-hydroxycholest-4-en-3-one;

[0015] iii) An enzyme that converts 7α-Hydroxycholest-4-en-3-one to 4-cholesten-7α,12α-diol-3-one;

[0016] iv) An enzyme that converts 4-cholesten-7α,12α-diol-3-one to 7α,12α-dihydroxy-5β-cholestan-3-one;

[0017] v) An enzyme that converts 7α,12α-dihydroxy-5β-cholestan-3-one to 3α,7α,12α-trihydroxy-5β-cholestane;

[0018] vi) An enzyme that converts 7α-Hydroxycholest-4-en-3-one to 7α-Hydroxy-5β-cholestan-3-one;

[0019] vii) an enzyme that converts 7α-hydroxy-5β-cholestan-3-one to 3α,7α-dihydroxy-5β-cholestane; and

[0020] viii) An enzyme that converts 3α,7α-dihydroxy-5β-cholestane to 3α,7α-dihydroxy-5β-cholestane via 3α,7α,26-trihydroxy-5β-cholestane and 3α,7α-dihydroxy-5β-cholestane-26-al into 3α,7α-dihydroxy-5β-cholestanate.

[0021] In the present invention, the genes encoding the enzymes can be operably linked in sequence according to the metabolic pathway for synthesizing the UDCA precursor.

[0022] In the present invention, the UDCA precursor is 7α-hydroxycholesterol, 7α-hydroxycholest-4-en-3-one, 4-cholesten-7α,12α-diol-3-one, 7α,12α-dihydroxy-5β-cholestane-3-one, 3α,7α,12α-trihydroxy-5β-cholestane, 3α,7α,12α,26-tetrahydroxy-5β-cholestane, 3α,7α,12α-trihydroxy-5β-cholestane-26-al, 3α,7α,12α-trihydroxy-5β-cholestanoate, (25R)-3α,7α,12α-trihydroxy-5β-cholestane-26-oil-CoA, (25S)-3α,7α,12α-trihydroxy-5β-cholestane-26-oleoyl-CoA, 3α,7α,12α-trihydroxy-5β-cholestane-24-enoyl-CoA, 3α,7α,12α,24-tetrahydroxy-5β-cholestanoyl-CoA, 3α,7α,12α,trihydroxy-5β-24-oxocholestanoyl-CoA, choleyl-CoA, 7α-hydroxy-5β-cholestane-3-one, 3α,7α-dihydroxy-5β-cholestane, 3α,7α,26-trihydroxy-5β-cholestane, 3α,7α-dihydroxy-5β-cholestane-26-al, 3α,7α-dihydroxy-5β-cholestanoyl-CoA, (25R)-3α,7α-dihydroxy-5β-cholestanoyl-CoA, (25S)-3α,7α-dihydroxy-5β-cholestanoyl-CoA, (24E)- 3α,7α-dihydroxy-5β-cholest-24-enoyl-CoA, 3α,7α,24-trihydroxy-5β-cholestanoyl-CoA, 3α,7α-dihydroxy-5β-24-oxocholestanoyl-CoA, chenodeoxycholoyl-CoA, cholic acid (CA) or chenodeoxycholic acid (CDCA).

[0023] In the present invention, the enzyme involved in the CA metabolic pathway among the enzymes may be at least one enzyme selected from the group consisting of i) to v).

[0024] In the present invention, the enzyme involved in the CDCA metabolic pathway among the enzymes may be at least one enzyme selected from the group consisting of i), ii), and vi) to viii).

[0025] In the present invention, the gene encoding the enzyme of i) may be CYP7A1 derived from human (Homo sapiens) or rabbit (Oryctolagus cuniculus).

[0026] In the present invention, the gene encoding the enzyme of ii) may be ERG26 derived from yeast (Saccharomyces cerevisiae), hsdD derived from Mycobacterium tuberculosis H37Rv, choD derived from Mycobacterium tuberculosis H37Rv, choB derived from Brevibacterium sterolicum, YKL107W derived from yeast (Saccharomyces cerevisiae), 3BHSD derived from Levilactobacillus brevis, transcript variant 1 of full-length form HSD3B7 derived from human (Homo sapiens), HSD3B7 derived from rabbit (Oryctolagus cuniculus), or HSD3B7 derived from cow (Bos Taurus).

[0027] In the present invention, the gene encoding the enzyme of iii) may be human (Homo sapiens) derived CYP8B1, rabbit (Oryctolagus cuniculus) derived CYP8B1, or chicken (Gallus gallus) derived CYP8B1.

[0028] In the present invention, the gene encoding the enzyme of iv) may be a transcript variant 1 of human (Homo sapiens) derived AKR1D1, bovine (Bos Taurus) derived AKR1D1, or chicken (Gallus gallus) derived AKR1D1.

[0029] In the present invention, the gene encoding the enzyme of v) may be AKR1C4 derived from Macaca fascicularis or hsdA derived from Comamonas testosteroni.

[0030] In the present invention, the gene encoding the enzyme of vi) may be a transcript variant 1 of human (Homo sapiens) derived AKR1D1, bovine (Bos Taurus) derived AKR1D1, or chicken (Gallus gallus) derived AKR1D1.

[0031] In the present invention, the genes encoding the enzyme of vii) and the enzyme of viii) may be human (Homo sapiens) derived AKR1C4 and human (Homo sapiens) derived CYP27A1, respectively.

[0032] In the present invention, the UPC2-1 gene may be additionally included.

[0033] In the present invention, the UPC2-1 gene may be derived from yeast.

[0034] In the present invention, a gene encoding the enzyme of the above i) and a gene encoding cytochrome p450 oxidoreductase (POR) and cytochrome b5 (CYB5) of homologous or heterologous origin may be additionally included.

[0035] In the present invention, the gene encoding the enzyme of i) may be derived from rabbit (Oryctolagus cuniculus), chicken (Gallus gallus), cow (Bos Taurus), or pig (Sus scrofa).

[0036] In the present invention, a gene encoding the enzyme of iii) above may additionally include a gene encoding cytochrome p450 oxidoreductase (POR) and cytochrome b5 (CYB5) of homologous or heterologous origin.

[0037] In the present invention, when the gene encoding the enzyme of iii) is from human (Homo sapiens), rabbit (Oryctolagus cuniculus) or chicken (Gallus gallus), a gene encoding cytochrome P450 oxidoreductase (POR) and cytochrome b5 (CYB5) of homologous origin may be additionally included.

[0038] In the present invention, when the gene encoding the enzyme of iii) is derived from rabbit (Oryctolagus cuniculus), a gene encoding a heterologous cytochrome p450 oxidoreductase (POR) and cytochrome b5 (CYB5) may be additionally included.

[0039] In the present invention, the gene encoding the enzyme of iii) and the gene encoding heterologous cytochrome p450 oxidoreductase (POR) and cytochrome b5 (CYB5) may be derived from brown rat (Rattus norvegicus), cow (Bos Taurus), or chicken (Gallus gallus).

[0040] In the present invention, the genes encoding cytochrome p450 oxidoreductase (POR) and cytochrome b5 (CYB5) can be expressed simultaneously with the genes encoding the enzymes of i) and / or iii).

[0041] In the present invention, a gene encoding adrenodoxin-NADP+ reductase (FDXR) may be additionally included.

[0042] In the present invention, the gene encoding the adrenodoxin-NADP+ reductase may be of human (Homo sapiens) origin.

[0043] In the present invention, the gene encoding the adrenodoxin-NADP+ reductase can be expressed simultaneously with the gene encoding the enzyme viii).

[0044] In the present invention, the gene encoding the enzyme of the above i) may include the nucleotide sequence of SEQ ID NO: 1 (human CYP7A1) or SEQ ID NO: 2 (rabbit CYP7A1).

[0045] In the present invention, the gene encoding the enzyme of ii) may include a nucleotide sequence of SEQ ID NO: 7 (transcriptional variant 1 of human full-length form HSD3B7), SEQ ID NO: 9 (rabbit HSD3B7), SEQ ID NO: 11 (bovine HSD3B7), SEQ ID NO: 13 (yeast ERG26), SEQ ID NO: 14 (Mycobacterium tuberculosis H37Rv hsdD), SEQ ID NO: 15 (Mycobacterium tuberculosis H37Rv choD), SEQ ID NO: 18 (yeast YKL107W), SEQ ID NO: 19 (Leviractobacillus brevis3BHSD), transcriptional variant 1 of human full-length form HSD3B7, specific sequence numbers of rabbit HSD3B7 or bovine HSD3B7).

[0046] In the present invention, the gene encoding the enzyme of iii) may include the nucleotide sequence of SEQ ID NO: 20 (human CYP8B1), SEQ ID NO: 22 (rabbit CYP8B1), or SEQ ID NO: 24 (chicken CYP8B1).

[0047] In the present invention, the gene encoding the enzyme of iv) may include the nucleotide sequence of SEQ ID NO: 26 (transcript variant 1 of human AKR1D1), SEQ ID NO: 27 (bovine AKR1D1), or SEQ ID NO: 31 (chicken AKR1D1).

[0048] In the present invention, the gene encoding the enzyme of v) may include the nucleotide sequence of SEQ ID NO: 35 (monkey AKR1C4) or SEQ ID NO: 39 (Comamonas testosterone hsdA).

[0049] In the present invention, the gene encoding the enzyme of vi) may include the nucleotide sequence of SEQ ID NO: 26 (human AKR1D1), SEQ ID NO: 27 (bovine AKR1D1), or SEQ ID NO: 31 (chicken AKR1D1).

[0050] In the present invention, the gene encoding the enzyme of vii) may include the nucleotide sequence of SEQ ID NO: 34.

[0051] In the present invention, the gene encoding the enzyme of viii) may include the nucleotide sequence of SEQ ID NO: 40.

[0052] In the present invention, the UPC2-1 gene may include the nucleotide sequence of SEQ ID NO: 55 (yeast UPC2-1).

[0053] In the present invention, the gene encoding the enzyme of the above i) and the gene encoding cytochrome p450 oxidoreductase (POR) of homologous or heterologous origin may include the nucleotide sequence of SEQ ID NO: 56 (human cytochrome P450 oxidoreductase), and the gene encoding cytochrome b5 (CYB5) of homologous or heterologous origin may include the nucleotide sequence of SEQ ID NO: 61 (human cytochrome b5).

[0054] In the present invention, the gene encoding the enzyme of iii) and the gene encoding cytochrome p450 oxidoreductase (POR) of homologous or heterologous origin may include the nucleotide sequence of SEQ ID NO: 66 (human cytochrome P450 oxidoreductase), and the gene encoding cytochrome b5 (CYB5) of homologous or heterologous origin may include the nucleotide sequence of SEQ ID NO: 71 (human cytochrome b5).

[0055] In the present invention, the gene encoding the adrenodoxin-NADP+ reductase may include the nucleotide sequence of SEQ ID NO: 76 (human FDXR).

[0056] In the present invention, the cell may be a yeast cell.

[0057] In the present invention, the yeast may be Saccharomyces cerevisiae.

[0058] The present invention also provides a method for producing a UDCA precursor using the aforementioned cells.

[0059] In the present invention, the precursor is 7α-hydroxycholesterol, 7α-hydroxycholest-4-en-3-one, 4-cholesten-7α,12α-diol-3-one, 7α,12α-dihydroxy-5β-cholestane-3-one, 3α,7α,12α-trihydroxy-5β-cholestane, 3α,7α,12α,26-tetrahydroxy-5β-cholestane, 3α,7α,12α-trihydroxy-5β-cholestane-26-al, 3α,7α,12α-trihydroxy-5β-cholestanoate, (25R)-3α,7α,12α-trihydroxy-5β-cholestane-26-oil-CoA, (25S)-3α,7α,12α-trihydroxy-5β-cholestane-26-oleoyl-CoA, 3α,7α,12α-trihydroxy-5β-cholestane-24-enoyl-CoA, 3α,7α,12α,24-tetrahydroxy-5β-cholestanoyl-CoA, 3α,7α,12α,trihydroxy-5β-24-oxocholestanoyl-CoA, choleyl-CoA, 7α-hydroxy-5β-cholestane-3-one, 3α,7α-dihydroxy-5β-cholestane, 3α,7α,26-trihydroxy-5β-cholestane, 3α,7α-dihydroxy-5β-cholestane-26-al, 3α,7α-dihydroxy-5β-cholestanoyl-CoA, (25R)-3α,7α-dihydroxy-5β-cholestanoyl-CoA, (25S)-3α,7α-dihydroxy-5β-cholestanoyl-CoA, (24E)- 3α,7α-dihydroxy-5β-cholest-24-enoyl-CoA, 3α,7α,24-trihydroxy-5β-cholestanoyl-CoA, 3α,7α-dihydroxy-5β-24-oxocholestanoyl-CoA, chenodeoxycholoyl-CoA, CA or CDCA.

[0060] The present invention establishes a biosynthetic pathway capable of continuously producing a UDCA precursor by introducing genes encoding a substance converting enzyme and / or a redox partner enzyme involved in the biosynthetic pathway of a UDCA precursor into yeast using a combination of genetic resources of various origins, thereby producing a UDCA precursor in an environmentally friendly and more stable manner compared to the conventional method of chemically synthesizing UDCA from animal-derived sources.

[0061] Figure 1a shows the metabolic pathway of CA, and Figure 1b shows the metabolic pathway of CDCA.

[0062] Figure 2 is the nucleotide sequence of the pYES2 vector.

[0063] Figure 3a is the nucleotide sequence of the pY-sHsCYP7A1 vector, Figure 3b is the nucleotide sequence of the sHsPOR gene expression cassette, Figure 3c is the nucleotide sequence of the sScNcp1 gene expression cassette, Figure 3d is the nucleotide sequence of the sHsCYB5 gene expression cassette, and Figure 3e is the nucleotide sequence of the sScUPC2-1 gene expression cassette.

[0064] Figure 4a shows an overview of plasmid construction of pY-sCP, pY-sCN, and pY-sCU, Figure 4b shows an overview of plasmid construction of pY-sCPB and pY-sCPU, Figure 4c shows an overview of plasmid construction of pY-sCNB and pY-sCNU, Figure 4d shows an overview of plasmid construction of pY-sCPBU and pY-sCNBU, and Figure 4e shows a schematic diagram of expression plasmid construction for conversion of 7α-hydroxycholesterol in yeast.

[0065] Figures 5a and 5b are graphs showing cell growth of a 7α-hydroxycholesterol production plasmid in the S. cerevisiae Y2805 strain.

[0066] Figure 6a is a graph showing cell growth according to cholesterol concentration in the S. cerevisiae BY4741 strain, and Figure 6b compares cell growth curves between an empty vector and a 7α-hydroxycholesterol production plasmid in the S. cerevisiae BY4741 strain.

[0067] Figure 7a is the nucleotide sequence of CYP7A1 derived from O. cuniculus, Figure 7b is the nucleotide sequence of POR (Cytochrome P450 oxidoreductase) derived from O. cuniculus, Figure 7c is the nucleotide sequence of CYB5 (Cytochrome b5) derived from O. cuniculus, Figure 7d is the nucleotide sequence of CYP7A1 derived from G. gallus, Figure 7e is the nucleotide sequence of POR (Cytochrome P450 oxidoreductase) derived from G. gallus, Figure 7f is the nucleotide sequence of CYB5 (Cytochrome b5) derived from G. gallus, Figure 7g is the nucleotide sequence of CYP7A1 derived from B. taurus, and Figure 7h is the nucleotide sequence of CYP7A1 derived from B. Figure 7i is the nucleotide sequence of B. Taurus-derived POR (Cytochrome P450 oxidoreductase), Figure 7j is the nucleotide sequence of B. Taurus-derived CYB5 (Cytochrome b5), Figure 7k is the nucleotide sequence of B. scrofa-derived CYP7A1, Figure 7k is the nucleotide sequence of B. scrofa-derived POR (Cytochrome P450 oxidoreductase), and Figure 7l is the nucleotide sequence of B. scrofa-derived CYB5 (Cytochrome b5).

[0068] Figure 8a shows a schematic diagram of plasmid construction of pY-sOcCP, pY-sGgCP, pY-sBtCP, and pY-sSsCP, Figure 8b shows a schematic diagram of plasmid construction of pY-sOcCPB, pY-sGgCPB, pY-sBtCPB, and pY-sSsCPB, and Figure 8c shows a schematic diagram of plasmid construction of pY-sOcCPBU, pY-sGgCPBU, pY-sBtCPBU, and pY-sSsCPBU.

[0069] Figure 9 shows the DNA fragmentation results of pY-sOcCPBU, pY-sGgCPBU, pY-sBtCPBU, and pY-sScCPBU using SacII, PacI, and SpeI, showing (A) 1 kb DNA ladder, (B) pY-sOCCPBU, (C) pY-sGgCPBU, (D) pY-sBtCPBU, and (E) pY-sSsCPBU.

[0070] Figures 10a to 10c are graphs showing cell growth of 7α-hydroxycholesterol production plasmid (I), 7α-hydroxycholesterol production plasmid (II), and 7α-hydroxycholesterol production plasmid (III), respectively, in that order.

[0071] Figure 11 is the nucleotide sequence of ERG26 derived from S. cerevisiae.

[0072] Figure 12 shows a schematic diagram of pY-sCPBUE plasmid construction.

[0073] Figure 13 shows the DNA fragmentation results of pY-sCPBUE using NheI and XhoI.

[0074] Figure 14a is a graph showing the effect of ERG26 overexpression on cell growth I, Figure 14b is a graph showing the effect of ERG26 overexpression on production of substances in the second stage of the CA pathway, Figure 14c is a graph showing the effect of ERG26 overexpression on cell growth II, and Figure 14d is a graph showing the effect of ERG26 overexpression on production of substances in the third stage of the CA pathway.

[0075] Figure 15a is the nucleotide sequence of the synthetic HSD3B3 gene (full form) derived from H. sapiens, Figure 15b is the nucleotide sequence of the synthetic HSD3B3 gene derived from R. norvegicus, Figure 15c is the nucleotide sequence of the synthetic HSD3B3 gene derived from O. cuniculus, Figure 15d is the nucleotide sequence of the synthetic HSD3B3 gene derived from U. maritimus, Figure 15e is the nucleotide sequence of the synthetic HSD3B3 gene derived from B. Taurus, and Figure 15f is the nucleotide sequence of the synthetic HSD3B3 gene derived from G. gallus.

[0076] Figure 16 is a schematic diagram of the plasmid construction of pY-sCPBUD(Hv), pY-sCPBUD(R), pY-sCPBUD(O), pY-sCPBUD(U), pY-sCPBUD(B), and pY-sCPBUD(G).

[0077] Figure 17 shows the DNA fragmentation results of pY-sCPBUD(Hv), pY-sCPBUD(R), pY-sCPBUD(O), pY-sCPBUD(U), pY-sCPBUD(B), and pY-sCPBUD(G) using ClaI, showing (A) 1 kb DNA ladder, (B) pY-sCPBUD(Hv), (C) pY-sCPBUD(R), (D) pY-sCPBUD(O), (E) 1 kb DNA ladder, (F) pY-sCPBUD(U), (G) pY-sCPBUD(B), (H) pY-sCPBUD(G).

[0078] Figure 18a is a graph showing the cell growth of the 7α-hydroxy-4-cholesten-3-one producing plasmid (average of the first and second batch results), Figure 18b is a graph showing the CA pathway ~2 step conversion material productivity of the candidate strain (average of the first and second batch results), Figure 18c is a graph showing the cell growth of the 7α-hydroxy-4-cholesten-3-one producing plasmid (average of the third batch results), and Figure 18d is a graph showing the CA pathway ~2 step conversion material productivity of the candidate strain (average of the first, second, and third batch results).

[0079] Figure 19 illustrates an enzymatic process for the production of 7α-hydroxy-4-cholesten-3-one from 7α-hydroxycholesterol.

[0080] Figure 20a is the nucleotide sequence of the synthetic hsdD gene derived from M. tuberculosis H37Rv, Figure 20b is the nucleotide sequence of the synthetic choD gene derived from M. tuberculosis H37Rv, Figure 20c is the nucleotide sequence of the synthetic choB gene derived from B. sterolicum, Figure 20d is the nucleotide sequence of the synthetic choB (w / o signal peptide) gene derived from B. sterolicum, Figure 2e is the nucleotide sequence of the synthetic YKL107W gene derived from S. cerevisiae, and Figure 20f is the nucleotide sequence of the synthetic 3BHSD gene derived from L. brevis.

[0081] Figure 21 is a schematic diagram of plasmid construction of pY-sCPBUMhD, pY-sCPBUMcD, pY-sCPBUBcB, pY-sCPBUBcB(woSS), pY-sCPBUSsR, and pY-sCPBULhD.

[0082] Figure 22 shows the DNA fragmentation results of pY-sCPBUMhD, pY-sCPBUMcD, pY-sCPBUBcB, pY-sCPBUBcB(woSS), pY-sCPBUSsR, and pY-sCPBULhD using restriction enzymes, showing (A) 1 kb DNA ladder, (B) pY-sCPBUMhD, (C) pY-sCPBUMcD, (D) pY-sCPBUBcB, (E) 1 kb DNA ladder, (F) pY-sCPBUBcB(woSS), (G) pY-sCPBUSsR, and (H) pY-sCPBULhD.

[0083] Figure 23a is a graph showing cell growth of a candidate plasmid producing 7α-hydroxy-4-cholesten-3-one.

[0084] Figure 23b is a graph showing the cholesterol and 7α-hydroxycholesterol productivity of the candidate strains (average of the results of the first and second batches).

[0085] Figure 23c is a graph showing the 7α-hydroxy-4-cholesten-3-one and 5-cholesten-3-one productivity of the candidate strain (average of the results of the first and second batches).

[0086] Figure 24 is a graph showing the cell growth of a candidate strain for producing a CA pathway ~3 step conversion material.

[0087] Figure 25a is the nucleotide sequence of the synthetic CYP8B1 gene derived from R. norvegicus, Figure 25b is the nucleotide sequence of the synthetic CYP8B1 gene derived from O. cuniculus, Figure 25c is the nucleotide sequence of the synthetic CYP8B1 gene derived from B. Taurus, Figure 25d is the nucleotide sequence of the synthetic CYP8B1 gene derived from G. gallus, and Figure 25e is the nucleotide sequence of the synthetic CYP8B1 gene derived from H. sapiens.

[0088] Figure 26a is the nucleotide sequence of a synthetic POR (Cytochrome P450 oxidoreductase) gene derived from R. norvegicus, Figure 26b is the nucleotide sequence of a synthetic POR (Cytochrome P450 oxidoreductase) gene derived from B. Taurus, Figure 26c is the nucleotide sequence of a synthetic POR (Cytochrome P450 oxidoreductase) gene derived from G. gallus, Figure 26d is the nucleotide sequence of a synthetic POR (Cytochrome P450 oxidoreductase) gene derived from H. sapiens, and Figure 26e is the nucleotide sequence of a synthetic POR (Cytochrome P450 oxidoreductase) gene derived from O. cuniculus.

[0089] Figure 27a is the nucleotide sequence of the synthetic CYB5 (Cytochrome b5) gene derived from R. norvegicus, Figure 27b is the nucleotide sequence of the synthetic CYB5 (Cytochrome b5) gene derived from O. cuniculus, Figure 27c is the nucleotide sequence of the synthetic CYB5 (Cytochrome b5) gene derived from B. Taurus, Figure 27d is the nucleotide sequence of the synthetic CYB5 (Cytochrome b5) gene derived from G. gallus, and Figure 27e is the nucleotide sequence of the synthetic CYB5 (Cytochrome b5) gene derived from H. sapiens.

[0090] Figure 28a shows a schematic diagram of 10 plasmid constructions for introduction of CA pathway step 3 genes, Figure 28b shows a schematic diagram of 3 plasmid constructions for introduction of POR (Cytochrome P450 oxidoreductase) genes, Figure 28c shows a schematic diagram of pY-sOcPOR plasmid construction into which O. cuniculus-derived POR (Cytochrome P450 oxidoreductase) genes are inserted, and Figure 28d shows a schematic diagram of 4 plasmid constructions for introduction of CYB5 (Cytochrome b5) genes.

[0091] Figure 29 shows the DNA fragmentation results of the redox partner gene introduction plasmids, (A) 1 kb DNA ladder, (B) pYH-sRnPOR, (C) pYH-sBtPOR, (D) pYH-sGgPOR, (E) 1 kb DNA ladder, (F) pYH-sOcPH, (G) pYH-sRnPH, (H) pYH-sBtPH, (I) pYH-sGgPH.

[0092] Figure 30a is a graph showing the average cell growth of a candidate plasmid (homologous gene combination) for producing 7α,12α-dihydroxycholest-4-en-3-one, and Figure 30b is a graph showing the average cell growth of a candidate plasmid (heterologous gene combination) for producing 7α,12α-dihydroxycholest-4-en-3-one.

[0093] Figure 31a is the nucleotide sequence of the synthetic AKR1D1 gene (transcript variant 2) derived from H. sapiens, Figure 31b is the nucleotide sequence of the synthetic AKR1D1 gene (transcript variant 1) derived from H. sapiens, Figure 31c is the nucleotide sequence of the synthetic AKR1D1 gene derived from B. Taurus, Figure 31d is the nucleotide sequence of the synthetic AKR1D1 gene derived from S. scrofa, Figure 31e is the nucleotide sequence of the synthetic AKR1D1 gene derived from O. cuniculus, and Figure 31f is the nucleotide sequence of the synthetic AKR1D1 gene derived from R. Figure 31g is the nucleotide sequence of the synthetic AKR1D1 gene derived from G. norvegicus, Figure 31h is the nucleotide sequence of the synthetic AKR1D1 gene derived from X. laevis, and Figure 31i is the nucleotide sequence of the synthetic AKR1D1 gene derived from F. verticillioides.

[0094] Figure 32a shows a schematic diagram of nine plasmid constructions for the production of CA pathway step 4 materials using pYH-sOcPH, and Figure 32b shows a schematic diagram of three plasmid constructions for the production of CA pathway step 4 materials using pYH-sRnPH.

[0095] Figure 33 shows the DNA fragmentation results of the 4-step gene introduction plasmid, (A) 1 kb DNA ladder, (B) pYH-sOcPHD(H), (C) pYH-sOcPHD(Hv), (D) pYH-sOcPHD(B), (E) pYH-sOcPHD(S), (F) 1 kb DNA ladder, (G) pYH-sOcPHD(O), (H) pYH-sOcPHD(R), (I) pYH-sOcPHD(G), (J) pYH-sOcPHD(X), (K) 1 kb DNA ladder, (L) pYH-sOcPHD(F), (M) pYH-sRnPHD(Hv), (N) pYH-sRnPHD(B), (O) pYH-sRnPHD(G) is shown.

[0096] Figure 34a is a graph showing the average cell growth results of a candidate plasmid for producing 7α,12α-dihydroxy-5β-cholestan-3-one using pYH-sOcPH, and Figure 34b is a graph showing the average cell growth results of a candidate plasmid for producing 7α,12α-dihydroxy-5β-cholestan-3-one using pYH-sRnPH.

[0097] Figure 35a is the nucleotide sequence of the synthetic AKR1C4 gene derived from H. sapiens, Figure 35b is the nucleotide sequence of the synthetic AKR1C4 gene derived from M. fascicularis, Figure 35c is the nucleotide sequence of the synthetic AKR1C4 gene derived from S. boliviensis, Figure 35d is the nucleotide sequence of the synthetic AKR1D1 gene derived from N. leucogenys, Figure 35e is the nucleotide sequence of the synthetic AKR1C4 gene derived from G. g. gorilla, and Figure 35f is the nucleotide sequence of the synthetic hsdA gene derived from C. testosteroni.

[0098] Figure 36a shows a schematic diagram of six plasmid constructions for the production of CA pathway step 5 materials using pYH-sOcPHD(Hv), Figure 36b shows a schematic diagram of six plasmid constructions for the production of CA pathway step 5 materials using pYH-sOcPHD(B), and Figure 36c shows a schematic diagram of one plasmid construction for the production of CA pathway step 5 materials using pYH-sRnPHD(B).

[0099] Figure 37a shows the DNA fragmentation results of the CA pathway step 5 genes introduced into pYH-sOcPHD(Hv), (M) 1 kb DNA ladder, (A) pYH-sOcPHDC(HvH), (B) pYH-sOcPHDC(HvM), (C) pYH-sOcPHDC(HvSb), (D) pYH-sOcPHDC(HvN), (E) pYH-sOcPHDC(HvGg), (F) pYH-sOcPHDC(HvC).

[0100] Figure 37b shows the DNA fragmentation results of the CA pathway step 5 genes introduced into pYH-sOcPHD(B), (M) 1 kb DNA ladder, (A) pYH-sOcPHDC(BH), (B) pYH-sOcPHDC(BM), (C) pYH-sOcPHDC(BSb), (D) pYH-sOcPHDC(BN), (E) pYH-sOcPHDC(BGg), (F) pYH-sOcPHDC(BC).

[0101] Figure 37c shows the DNA fragmentation results of pYH-sRnPHDC(BC), showing (M) 1 kb DNA ladder and (A) pYH-sRnPHDC(BC).

[0102] Figure 38a is a graph showing the average cell growth results of candidate plasmids for producing 3α,7α,12α-trihydroxy-5β-cholestane using pYH-sOcPHD(HV), pYH-sOcPHD(B), and pYH-sRnPHD(B), and Figure 38b is a graph showing the average cell growth results of candidate plasmids for producing 3α,7α,12α-trihydroxy-5β-cholestane using pYH-sOcPHD(HV), pYH-sOcPHD(B), and pYH-sRnPHD(B).

[0103] Figure 39a is the nucleotide sequence of the synthetic AKR1D1 gene (transcript variant 1) derived from H. sapiens, Figure 39b is the nucleotide sequence of the synthetic AKR1D1 gene derived from B. taurus, Figure 39c is the nucleotide sequence of the synthetic AKR1D1 gene derived from S. scrofa, Figure 39d is the nucleotide sequence of the synthetic AKR1D1 gene derived from O. cuniculus, Figure 39e is the nucleotide sequence of the synthetic AKR1D1 gene derived from R. norvegicus, Figure 39f is the nucleotide sequence of the synthetic AKR1D1 gene derived from G. gallus, Figure 39g is the nucleotide sequence of the synthetic AKR1D1 gene derived from X. laevis, and Figure 39h is the nucleotide sequence of the synthetic AKR1D1 gene derived from F. verticillioides-derived synthetic AKR1D1 gene, and Figure 39i is the nucleotide sequence of the H. sapiens-derived AKR1D1 gene (transcript variant 2).

[0104] Figure 40 is a schematic diagram of the construction of nine plasmids for the introduction of genes in the CDCA pathway step 3.

[0105] Figure 41 shows the fragmentation results of nine plasmids constructed by introducing the AKR1D1 gene, namely (A) 1 kb DNA ladder, (B) pYH-sHsAKR1D1, (C) pYH-sHsAKR1D1(v1), (D) pYH-sBtAKR1D1, (E) 1 kb DNA ladder, (F) pYH-sSsAKR1D1, (G) pYH-sOcAKR1D1, (H) pYH-sRnAKR1D1, (I) 1 kb DNA ladder, (J) pYH-sGgAKR1D1, (K) pYH-sXlAKR1D1, and (L) pYH-sFvAKR1D1.

[0106] Figure 42a is the nucleotide sequence of the synthetic AKR1C4 gene derived from H. sapiens, Figure 42b is the nucleotide sequence of the CYP27A1 gene derived from H. sapiens, and Figure 42c is the nucleotide sequence of the FDXR gene derived from H. sapiens.

[0107] Figure 43a is a schematic diagram of plasmid construction of pYH-sHsDC(HvH) and pYH-sBtDC(H), Figure 43b is a schematic diagram of plasmid construction of pYH-sHsDCC(HvHH) and pYH-sBtDCC (HH), and Figure 43c is a schematic diagram of plasmid construction of pYH-sHsDCChF(HvHH) and pYH-sBtDCChF(HH).

[0108] Figure 44a shows the DNA fragmentation results of pYH-sHsDC(HvH) and pYH-sBtDC(H) using PstI and SacI, showing (A) 1 kb DNA ladder, (B) marker, (C) pYH-sHsDC(HvH) (uncut), (D) pYH-sHsDC(HvH) (cut, PstI), (E) marker, (F) pYH-sBtDC(H) (uncut), and (G) pYH-sBtDC(H) (cut, SacI).

[0109] Figure 44b shows the fragmentation results of pYH-sHsDCC(HvHH) and pYH-sBtDCC(HH) using NdeI, showing (A) 1 kb DNA ladder, (B) marker, (C) pYH-sHsDCC(HvHH) (uncut), (D) pYH-sHsDCC(HvHH) (cut), (E) marker, (F) pYH-sBtDCC(HH) (uncut), and (G) pYH-sBtDCC(HH) (cut).

[0110] Figure 44c shows the DNA fragmentation results of pYH-sHsDCChF(HvHH) and pYH-sBtDCChF(HH) using NdeI, showing (A) 1 kb DNA ladder, (B) marker, (C) pYH-sHsDCChF(HvHH) (uncut), (D) pYH-sHsDCChF(HvHH) (cut), (E) marker, (F) pYH-sBtDCChF(HH) (uncut), (G) pYH-sBtDCChF(HH) (cut).

[0111] Figure 45 is a graph showing cell growth of the CDCA pathway step 3, 4, and 7 material production plasmids.

[0112] Hereinafter, the present invention will be described in more detail.

[0113] All technical terms used in this invention, unless otherwise defined, have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of this invention.

[0114] As described above, the existing method of synthesizing UDCA has problems such as difficulty in stable supply of raw materials for UDCA synthesis due to environmental issues, low yield, and risk of reaction process, and therefore, there is a need for the development of a new technology that can stably produce raw materials using environmentally friendly biotechnology. Accordingly, the inventors of the present invention sought a solution to the above-mentioned problem by introducing various microbial or mammalian-derived genetic resources into yeast to establish a biosynthetic pathway of UDCA precursors and to secure cells that stably produce UDCA precursors in an environmentally friendly manner. The genetically modified cell producing the UDCA precursor according to the present invention includes at least one gene encoding an enzyme involved in the metabolic pathway for synthesizing UDCA, and thus can produce the UDCA precursor in an environmentally friendly and more stable manner.

[0115] Accordingly, the present invention relates to a genetically modified cell producing a UDCA precursor comprising at least one gene encoding an enzyme involved in a metabolic pathway for synthesizing a UDCA precursor from cholesterol and a recombinant vector comprising at least one gene encoding an enzyme involved in a metabolic pathway for synthesizing a UDCA precursor from cholesterol.

[0116] In the present invention, the enzyme involved in the metabolic pathway for synthesizing the UDCA precursor from the cholesterol may be any one or more of the following enzymes i) to viii), but is not limited thereto:

[0117] i) An enzyme that converts cholesterol into 7α-hydroxycholesterol;

[0118] ii) An enzyme that converts 7α-hydroxycholesterol to 7α-hydroxycholest-4-en-3-one;

[0119] iii) An enzyme that converts 7α-Hydroxycholest-4-en-3-one to 4-cholesten-7α,12α-diol-3-one;

[0120] iv) An enzyme that converts 4-cholesten-7α,12α-diol-3-one to 7α,12α-dihydroxy-5β-cholestan-3-one;

[0121] v) An enzyme that converts 7α,12α-dihydroxy-5β-cholestan-3-one to 3α,7α,12α-trihydroxy-5β-cholestane;

[0122] vi) An enzyme that converts 7α-Hydroxycholest-4-en-3-one to 7α-Hydroxy-5β-cholestan-3-one;

[0123] vii) an enzyme that converts 7α-hydroxy-5β-cholestan-3-one to 3α,7α-dihydroxy-5β-cholestane; and

[0124] viii) An enzyme that converts 3α,7α-dihydroxy-5β-cholestane to 3α,7α-dihydroxy-5β-cholestane via 3α,7α,26-trihydroxy-5β-cholestane and 3α,7α-dihydroxy-5β-cholestane-26-al into 3α,7α-dihydroxy-5β-cholestanate.

[0125] In the present invention, the genes encoding the enzymes can be operably linked in sequence according to the metabolic pathway for synthesizing the UDCA precursor.

[0126] In the present invention, the UDCA precursor may be included without limitation as long as it is a substance produced by the involvement of one or more enzymes among i) to viii) in the metabolic pathway for synthesizing the UDCA precursor from cholesterol. For example, 7α-hydroxycholesterol, 7α-hydroxycholest-4-en-3-one, 4-cholesten-7α,12α-diol-3-one, 7α,12α-dihydroxy-5β-cholestane-3-one, 3α,7α,12α-trihydroxy-5β-cholestane, 3α,7α,12α,26-tetrahydroxy-5β-cholestane, 3α,7α,12α-trihydroxy-5β-cholestane-26-al, 3α,7α,12α-trihydroxy-5β-cholestanoate, (25R)-3α,7α,12α-trihydroxy-5β-cholestane-26-oil-CoA, (25S)-3α,7α,12α-trihydroxy-5β-cholestane-26-oleoyl-CoA, 3α,7α,12α-trihydroxy-5β-cholestane-24-enoyl-CoA, 3α,7α,12α,24-tetrahydroxy-5β-cholestanoyl-CoA, 3α,7α,12α,trihydroxy-5β-24-oxocholestanoyl-CoA, choleyl-CoA, 7α-hydroxy-5β-cholestane-3-one, 3α,7α-dihydroxy-5β-cholestane, 3α,7α,26-trihydroxy-5β-cholestane, 3α,7α-dihydroxy-5β-cholestane-26-al, 3α,7α-dihydroxy-5β-cholestanoyl-CoA, (25R)-3α,7α-dihydroxy-5β-cholestanoyl-CoA, (25S)-3α,7α-dihydroxy-5β-cholestanoyl-CoA, (24E)-3α,7α-dihydroxy-5β-cholest-24-enoyl-CoA, 3α,7α,24-trihydroxy-5β-cholestanoyl-CoA, 3α,7α-dihydroxy-5β-24-oxocholestanoyl-CoA, chenodeoxycholoyl-CoA, cholic acid (CA), or chenodeoxycholic acid (CDCA), but is not limited thereto.

[0127] When the genetically modified cell producing the UDCA precursor according to the present invention comprises a gene encoding an enzyme involved in the CA metabolic pathway, the cell may comprise at least one gene encoding an enzyme selected from the group consisting of i) to v).

[0128] First, the enzyme of the above i) is an enzyme that produces 7α-hydroxycholesterol (7HC) using cholesterol as a substrate, and the gene encoding it can be derived from various mammals, for example, the mammal can be a human (Homo sapiens), a rabbit (Oryctolagus cuniculus), a chicken (Gallus gallus), a cow (Bos Taurus), or a pig (Sus scrofa), but is not limited thereto. Preferably, the mammal can be a human (Homo sapiens), a rabbit (Oryctolagus cuniculus), a cow (Bos Taurus), or a pig (Sus scrofa), and more preferably, the mammal can be a human (Homo sapiens) or a rabbit (Oryctolagus cuniculus).

[0129] In the present invention, it is preferable that the gene encoding the enzyme of i) is expressed in combination with the enzyme of i) and the redox partner enzymes POR (Cytochrome P450 oxidoreductase) and CYB5 genes and the cholesterol absorption gene UPC2-1 gene, taking into account the productivity of 7α-hydroxycholesterol. At this time, the POR (Cytochrome P450 oxidoreductase) gene and the CYB5 (Cytochrome b5) gene may be of homologous or heterologous origin to the gene encoding the enzyme of i). Preferably, the POR (Cytochrome P450 oxidoreductase) gene and the CYB5 (Cytochrome b5) gene may be derived from various mammals, for example, but not limited to, human (Homo sapiens), rabbit (Oryctolagus cuniculus), chicken (Gallus gallus), cow (Bos Taurus), or pig (Sus scrofa). The above UPC2-1 gene may be derived from yeast (Saccharomyces cerevisiae), but is not limited thereto.

[0130] Second, the enzyme of the above ii) is an enzyme that produces 7α-hydroxycholesterol as a substrate to produce 7α-hydroxycholesterol-4-en-3-one, and the gene encoding it can be derived from various mammals or microorganisms. Examples of the mammals include, but are not limited to, humans (Homo sapiens), mice (Rattus norvegicus), rabbits (Oryctolagus cuniculus), bears (Ursus maritimus), cows (Bos Taurus), or chickens (Gallus gallus). Preferably, the mammals may be humans (Homo sapiens), rabbits (Oryctolagus cuniculus), or cows (Bos Taurus). Examples of the above microorganisms include, but are not limited to, Mycobacterium tuberculosis, Brevibacterium sterolicum, Saccharomyces cerevisiae, and Levilactobacillus brevis. Preferably, the mammal may be Saccharomyces cerevisiae, Mycobacterium tuberculosis, or Levilactobacillus brevis.

[0131] Specifically, when the gene encoding the enzyme of the above ii) is a microorganism, the gene encoding the enzyme of the above ii) may be ERG26 derived from yeast (Saccharomyces cerevisiae), hsdD derived from Mycobacterium tuberculosis H37Rv, choD derived from Mycobacterium tuberculosis H37Rv, choB derived from Brevibacterium sterolicum, YKL107W derived from yeast (Saccharomyces cerevisiae), or 3BHSD derived from Levilactobacillus brevis.

[0132] Thirdly, the enzyme of the above iii) is an enzyme that produces 4-cholesten-7α,12α-diol-3-one using 7α-hydroxycholest-4-en-3-one as a substrate, and the gene encoding it can be derived from various mammals, for example, the mammal can be a human (Homo sapiens), a mouse (Rattus norvegicus), a rabbit (Oryctolagus cuniculus), a cow (Bos Taurus), or a chicken (Gallus gallus), but is not limited thereto. Preferably, the mammal can be a human (Homo sapiens), a rabbit (Oryctolagus cuniculus), or a chicken (Gallus gallus).

[0133] In the present invention, the gene encoding the enzyme of iii) is preferably expressed in combination with the enzyme of iii) and the redox partner enzymes POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5) genes, taking into account the productivity of 4-cholesten-7α,12α-diol-3-one. At this time, the POR (Cytochrome P450 oxidoreductase) gene and the CYB5 (Cytochrome b5) gene may be of homologous or heterologous origin, respectively, to the gene encoding the enzyme of iii). Preferably, the POR (Cytochrome P450 oxidoreductase) gene and the CYB5 (Cytochrome b5) gene may be derived from various mammals, for example, the mammal may be a human (Homo sapiens), a mouse (Rattus norvegicus), a rabbit (Oryctolagus cuniculus), a cow (Bos Taurus), or a chicken (Gallus gallus), but is not limited thereto.

[0134] According to one embodiment of the present invention, the gene encoding the enzyme of iii) can be expressed simultaneously with a gene encoding a homologous POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5), and at this time, the gene encoding the homologous POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5) may be, but is not limited to, a human (Homo sapiens), a brown rat (Rattus norvegicus), a rabbit (Oryctolagus cuniculus), a cow (Bos Taurus), or a chicken (Gallus gallus).

[0135] According to a preferred embodiment of the present invention, when the gene encoding the enzyme of iii) is from human (Homo sapiens), rabbit (Oryctolagus cuniculus) or chicken (Gallus gallus), a gene encoding POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5) of homologous origin may be additionally included.

[0136] Exceptionally, if the gene encoding the enzyme of the above iii) is from a brown rat (Rattus norvegicus) or a cow (Bos Taurus), it may not include the genes encoding POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5) of homologous origin.

[0137] According to one embodiment of the present invention, the gene encoding the enzyme of iii) may additionally include a gene encoding a heterologous POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5), and in this case, the gene encoding the homologous POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5) may be, but is not limited to, a human (Homo sapiens), a brown rat (Rattus norvegicus), a rabbit (Oryctolagus cuniculus), a cow (Bos Taurus), or a chicken (Gallus gallus).

[0138] According to a preferred embodiment of the present invention, when the gene encoding the enzyme of iii) is derived from a rabbit (Oryctolagus cuniculus), a gene encoding a heterologous POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5) may be additionally included, and in this case, the gene encoding the enzyme of iii) and the gene encoding a heterologous POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5) may be derived from a brown rat (Rattus norvegicus), a cow (Bos Taurus), or a chicken (Gallus gallus).

[0139] In the present invention, it is preferable that the gene encoding POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5) be expressed simultaneously with the gene encoding the enzyme of i) and / or the gene encoding the enzyme of iii).

[0140] Fourth, the enzyme of the above iv) is an enzyme that produces 7α,12α-dihydroxy-5β-cholestane-3-one using 4-cholestene-7α,12α-diol-3-one as a substrate, and the gene encoding the same may be derived from various mammals, amphibians or microorganisms, for example, the mammal may be a human (Homo sapiens), a brown rat (Rattus norvegicus), a rabbit (Oryctolagus cuniculus), a cow (Bos Taurus), a chicken (Gallus gallus) or a pig (Sus scrofa), but is not limited thereto. Preferably, the mammal may be a human (Homo sapiens), a cow (Bos Taurus) or a chicken (Gallus gallus). The amphibian may be Xenopus laevis, but is not limited thereto. The above microorganism may be, but is not limited to, Fusarium verticillioides.

[0141] Fifthly, the enzyme of the above v) is an enzyme that uses 7α,12α-dihydroxy-5β-cholestan-3-one as a substrate to produce 3α,7α,12α-trihydroxy-5β-cholestan, and the gene encoding the same may be derived from various mammals or microorganisms. For example, the mammal may be, but is not limited to, a human (Homo sapiens), a Macaca fascicularis, a squirrel monkey (Saimiri boliviensis boliviensis), a northern white-cheeked gibbon (Nomascus leucogenys), or a western lowland gorilla (Gorilla gorilla gorilla). Preferably, the mammal may be a Macaca fascicularis. The microorganism may be, but is not limited to, Comamonas testosteroni.

[0142] In the present invention, when the final product of the genetically modified cell producing the UDCA precursor is CA, the UDCA precursor is 7α-hydroxycholesterol, 7α-hydroxycholest-4-en-3-one, 4-cholesten-7α,12α-diol-3-one, 7α,12α-dihydroxy-5β-cholestan-3-one, 3α,7α,12α-trihydroxy-5β-cholestan, 3α,7α,12α,26-tetrahydroxy-5β-cholestan, 3α,7α,12α-trihydroxy-5β-cholestan-26-al, 3α,7α,12α-trihydroxy-5β-cholestanoate, (25R)-3α,7α,12α-trihydroxy-5β-cholestane-26-oleoyl-CoA, (25S)-3α,7α,12α-trihydroxy-5β-cholestane-26-oleoyl-CoA, 3α,7α,12α-trihydroxy-5β-cholestane-24-enoyl-CoA, 3α,7α,12α,24-tetrahydroxy-5β-cholestanoyl-CoA, 3α,7α,12α,trihydroxy-5β-24-oxocholestanoyl-CoA, choleyl-CoA or CA.

[0143] When the genetically modified cell producing the UDCA precursor according to the present invention comprises a gene encoding an enzyme involved in the CDCA metabolic pathway, the cell may comprise at least one gene encoding an enzyme selected from the group consisting of i), ii), and vi) to viii).

[0144] First, the enzyme of the above i) is an enzyme that uses cholesterol as a substrate to produce 7α-hydroxycholesterol (7HC), and since it is the same as the case where the final product of a genetically modified cell producing a UDCA precursor is CA, its description is omitted.

[0145] Second, the enzyme of the above ii) is an enzyme that produces 7α-hydroxycholesterol as a substrate to produce 7α-hydroxycholesterol-4-en-3-one, and is the same as when the final product of a genetically modified cell producing a UDCA precursor is CA, so its description is omitted.

[0146] Thirdly, the enzyme of vi) above is an enzyme that produces 7α-hydroxy-5β-cholestan-3-one using 7α-hydroxycholest-4-en-3-one as a substrate, and the gene encoding it can be derived from various mammals, amphibians or microorganisms, for example, the mammal can be a human (Homo sapiens), a brown rat (Rattus norvegicus), a rabbit (Oryctolagus cuniculus), a cow (Bos Taurus), a chicken (Gallus gallus) or a pig (Sus scrofa), but is not limited thereto. Preferably, the mammal can be a human (Homo sapiens), a cow (Bos Taurus) or a chicken (Gallus gallus). The amphibian can be Xenopus laevis, but is not limited thereto. The above microorganism may be, but is not limited to, Fusarium verticillioides.

[0147] Fourth, the enzyme of the above vii) is an enzyme that produces 3α,7α-dihydroxy-5β-cholestane using 7α-hydroxy-5β-cholestane-3-one as a substrate, and the gene encoding this may be derived from various mammals, and for example, the mammal may be a human (Homo sapiens), but is not limited thereto.

[0148] Fifthly, the enzyme of the above viii) is an enzyme that uses 3α,7α-dihydroxy-5β-cholestane as a substrate to produce 3α,7α,26-trihydroxy-5β-cholestane and 3α,7α-dihydroxy-5β-cholestane-26-al, and ultimately produces 3α,7α-dihydroxy-5β-cholestane. The gene encoding this may be derived from various mammals. For example, the mammal may be a human (Homo sapiens), but is not limited thereto.

[0149] In the present invention, it is preferable that the gene encoding the enzyme of viii) is expressed in combination with a gene encoding the enzyme of viii) and the redox partner enzyme, adrenodoxin-NADP+ reductase (FDXR), in consideration of the productivity of 3α,7α-dihydroxy-5β-cholestanate. At this time, the gene encoding the adrenodoxin-NADP+ reductase may be derived from various mammals, and for example, the mammal may be a human (Homo sapiens), but is not limited thereto.

[0150] In the present invention, the gene encoding the enzyme of i) may include any one of the nucleotide sequences of sequence numbers 1 to 5 described in Table 1 below.

[0151] [Table 1]

[0152]

[0153]

[0154]

[0155]

[0156] Preferably, the gene encoding the enzyme of the above i) may include the nucleotide sequence of SEQ ID NO: 1 (human CYP7A1) or SEQ ID NO: 2 (rabbit CYP7A1). In the present invention, the gene encoding the enzyme of the above ii) may include any one of the nucleotide sequences of SEQ ID NOs: 6 to 19 described in Table 2 below.

[0157] [Table 2]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165] Preferably, the gene encoding the enzyme of the above ii) may include a nucleotide sequence of SEQ ID NO: 7 (transcript variant 1 of human full-length form HSD3B7), SEQ ID NO: 9 (rabbit HSD3B7), SEQ ID NO: 11 (bovine HSD3B7), SEQ ID NO: 13 (yeast ERG26), SEQ ID NO: 14 (Mycobacterium tuberculosis H37Rv hsdD), SEQ ID NO: 15 (Mycobacterium tuberculosis H37Rv choD), SEQ ID NO: 18 (yeast YKL107W), SEQ ID NO: 19 (Lactobacillus brevis3BHSD).

[0166] In the present invention, the gene encoding the enzyme of iii) may include any one of the nucleotide sequences of SEQ ID NOs: 20 to 24 described in Table 3 below.

[0167] [Table 3]

[0168]

[0169]

[0170]

[0171] Preferably, the gene encoding the enzyme of the above iii) may include the nucleotide sequence of SEQ ID NO: 20 (human CYP8B1), SEQ ID NO: 22 (rabbit CYP8B1) or SEQ ID NO: 24 (chicken CYP8B1).

[0172] In the present invention, the gene encoding the enzyme of iv) may include any one of the nucleotide sequences of SEQ ID NOs: 25 to 33 described in Table 4 below.

[0173] [Table 4]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179] Preferably, the gene encoding the enzyme of the above iv) may include the nucleotide sequence of SEQ ID NO: 26 (transcript variant 1 of human AKR1D1), SEQ ID NO: 27 (bovine AKR1D1) or SEQ ID NO: 31 (chicken AKR1D1).

[0180] In the present invention, the gene encoding the enzyme of v) may include any one of the nucleotide sequences of SEQ ID NOs: 34 to 39 described in Table 5 below.

[0181] [Table 5]

[0182]

[0183]

[0184]

[0185]

[0186] Preferably, the gene encoding the enzyme of the above v) may comprise the nucleotide sequence of SEQ ID NO: 35 (monkey AKR1C4) or SEQ ID NO: 39 (Comamonas testosterone hsdA).

[0187] In the present invention, the gene encoding the enzyme of vi) may include any one of the nucleotide sequences of SEQ ID NOs: 26 to 33 described in Table 4.

[0188] Preferably, the gene encoding the enzyme of the above vi) may include the nucleotide sequence of SEQ ID NO: 26 (human AKR1D1).

[0189] In the present invention, the gene encoding the enzyme of vii) may include any one of the nucleotide sequences of SEQ ID NOs: 34 to 39 described in Table 5.

[0190] Preferably, the gene encoding the enzyme of the above vii) may include the nucleotide sequence of SEQ ID NO: 34 (human AKR1C4) of the above Table 5.

[0191] In the present invention, the gene encoding the enzyme of viii) may include any one of the nucleotide sequences of SEQ ID NOs: 40 to 54 of Table 6 below.

[0192] [Table 6]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204] Preferably, the gene encoding the enzyme of the above viii) may include the nucleotide sequence of SEQ ID NO: 40 (human CYP27A1).

[0205] In the present invention, the cell may additionally include a UPC2-1 gene, which is a cholesterol absorption gene, and the UPC2-1 gene may be derived from yeast (Saccharomyces cerevisiae). The UPC2-1 gene may include the nucleotide sequence of the following SEQ ID NO: 55.

[0206] [Sequence number 55]

[0207]

[0208]

[0209] In the present invention, the gene encoding the enzyme of the above i) and the gene encoding POR (Cytochrome P450 oxidoreductase) of homologous or heterologous origin may include any one of the nucleotide sequences of SEQ ID NOs: 56 to 60 described in Table 7 below.

[0210] [Table 7]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217] Preferably, the gene encoding the enzyme of the above i) and the gene encoding POR (Cytochrome P450 oxidoreductase) of homologous or heterologous origin may include the nucleotide sequence of SEQ ID NO: 56 (human POR).

[0218] In the present invention, the gene encoding the enzyme of the above i) and the gene encoding CYB5 (Cytochrome b5) of homologous or heterologous origin may include any one of the nucleotide sequences of SEQ ID NOs: 61 to 65 described in Table 8 below.

[0219] [Table 8]

[0220]

[0221]

[0222] Preferably, the gene encoding the enzyme of the above i) and the gene encoding CYB5 (Cytochrome b5) of homologous or heterologous origin may include the nucleotide sequence of SEQ ID NO: 61 (human CYB5).

[0223] In the present invention, the gene encoding the enzyme of iii) above and the gene encoding POR (Cytochrome P450 oxidoreductase) of homologous or heterologous origin may include any one of the nucleotide sequences of SEQ ID NOs: 66 to 70 described in Table 9 below.

[0224] [Table 9]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230] Preferably, the gene encoding the enzyme of iii) above and the gene encoding POR (Cytochrome P450 oxidoreductase) of homologous or heterologous origin may include the nucleotide sequence of SEQ ID NO: 66 (human POR).

[0231] In the present invention, the gene encoding the enzyme of iii) above and the gene encoding CYB5 (Cytochrome b5) of homologous or heterologous origin may include any one of the nucleotide sequences of SEQ ID NOs: 71 to 75 described in Table 10 below.

[0232] [Table 10]

[0233]

[0234]

[0235] Preferably, the gene encoding the enzyme of the above iii) and the gene encoding CYB5 (Cytochrome b5) of homologous or heterologous origin may include the nucleotide sequence of SEQ ID NO: 71 (human CYB5).

[0236] In the present invention, the gene encoding the adrenodoxin-NADP+ reductase may include the nucleotide sequence of the following sequence numbers 76 to 90.

[0237] [Table 11]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248] Preferably, the gene encoding the enzyme of iii) above and the gene encoding adrenodoxin-NADP+ reductase of homologous or heterologous origin may include the nucleotide sequence of SEQ ID NO: 76 (human FDXR).

[0249] In the present invention, at least one gene encoding an enzyme involved in a metabolic pathway for synthesizing a UDCA precursor from cholesterol may each comprise or consist of a nucleotide sequence substantially identical to the nucleotide sequence designated by the above sequence number.

[0250] As used herein, the term "substantially identical" means that the sequence is at least 50% identical. In some instances, the term "substantially identical" refers to a sequence that is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a reference sequence.

[0251] In the present invention, the cell may be a yeast cell, and the yeast may be Saccharomyces cerevisiae.

[0252] The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Vectors include nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules comprising one or more free ends, nucleic acid molecules without free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and various other polynucleotides known in the art.

[0253] The term "recombinant vector" used herein refers to an expression vector of a target polypeptide (nucleic acid) that can express the target polypeptide with high efficiency in an appropriate host cell when the encoding gene of the target polypeptide to be expressed is operably linked, and the recombinant vector can be expressed in the host cell. The host cell may be a prokaryotic cell, and depending on the type of host cell, expression control sequences such as a promoter, terminator, enhancer, etc., sequences for membrane targeting or secretion, etc. can be appropriately selected and combined in various ways depending on the purpose.

[0254] The term “promoter”, “nucleic acid having promoter activity” or “promoter sequence” according to the present invention means a nucleic acid that is functionally linked to a nucleic acid to be transcribed and regulates transcription of the nucleic acid.

[0255] In this context, a "functional" or "operable" linkage means, for example, the sequential arrangement of one of the nucleic acids having promoter activity, the nucleic acid sequence to be transcribed, and any additional regulatory elements, for example, a nucleic acid sequence ensuring transcription of the nucleic acid, and for example, a terminator, such that each regulatory element can perform its function in the transcription of the nucleic acid sequence. This does not necessarily require a direct link in the chemical sense.

[0256] In addition to promoters and terminators, examples of other regulatory elements that may be mentioned include targeting sequences, enhancers, polyadenylation signals, selectable markers, amplification signals, origins of replication, etc.

[0257] Expression vectors can be stably or transiently introduced into host cells using established techniques, including but not limited to electroporation, calcium phosphate precipitation, DEAE-dextran-mediated transfection, liposome-mediated transfection, and heat shock. For stable transformation, the nucleic acid may generally additionally include a selectable marker, such as any of several well-known selectable markers, such as neomycin resistance, ampicillin resistance, tetracycline resistance, chloramphenicol resistance, kanamycin resistance, and the like. In some embodiments, the nucleic acid by which the host cell is genetically modified is an expression vector comprising a nucleic acid comprising a nucleotide sequence encoding a gene product, such as an enzyme, a transcription factor, or the like.

[0258] Suitable expression vectors include, but are not limited to, baculovirus vectors, bacteriophage vectors, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral vectors (e.g., vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40 herpes simplex virus, etc.), P1-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vector specific for a particular host of interest (e.g., yeast). Thus, for example, a nucleic acid encoding a gene product(s) is comprised in any one of a variety of expression vectors for expressing the gene product(s). Such vectors include chromosomal, non-chromosomal, and synthetic DNA sequences.

[0259] The present invention also relates to a method for producing a UDCA precursor using the above-described recombinant vector or a cell transformed with the above-described recombinant vector.

[0260] In the present invention, the description of the UDCA precursor is the same as described above, so its description is omitted.

[0261] In the present invention, the transformed cell is a genetically modified cell that produces a UDCA precursor, and since the description thereof is the same as described above, its description is omitted.

[0262] In the present invention, the transformed cells can be cultured under fermentation conditions suitable for converting the substrate cholesterol into a UDCA precursor. Suitable conditions are conditions under which the transformed cells (i.e., host cells) can maintain growth and / or survival and produce a UDCA precursor.

[0263] Non-limiting examples of suitable conditions include a suitable culture medium (e.g., a culture medium having a suitable nutrient content (e.g., a suitable carbon content, a suitable nitrogen content, a suitable phosphorus content), a suitable supplement content, a suitable trace metal content, a suitable pH), a suitable temperature, a suitable feed rate, a suitable pressure, a suitable oxygenation level, a suitable fermentation period, a suitable fermentation volume (i.e., the volume of culture medium containing the recombinant host cells), and a suitable fermentation vessel.

[0264] Suitable culture media include any culture medium that allows the host cells to grow and / or survive and produce UDCA precursors. Typically, the culture medium may be an aqueous medium containing, but is not limited to, a carbon source, an assimilable nitrogen source (i.e., a nitrogen-containing compound capable of releasing nitrogen in a form suitable for metabolic utilization by the host cells), and a phosphate source.

[0265] Non-limiting examples of carbon sources include monosaccharides, disaccharides, polysaccharides, acetate, ethanol, methanol, glycerol, methane, and combinations thereof. Non-limiting examples of monosaccharides include, but are not limited to, dextrose (glucose), fructose, galactose, xylose, arabinose, and combinations thereof. Non-limiting examples of disaccharides include, but are not limited to, sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of polysaccharides include, but are not limited to, starch, glycogen, cellulose, amylose, hemicellulose, maltodextrin, and combinations thereof.

[0266] Non-limiting examples of assimilable nitrogen sources include, but are not limited to, anhydrous ammonia, ammonium sulfate, ammonium hydroxide, ammonium nitrate, diammonium phosphate, monoammonium phosphate, ammonium pyrophosphate, ammonium chloride, sodium nitrate, urea, peptone, protein hydrolysates, corn steep liquor, corn steep solids, waste grain, waste grain extract, and yeast extract. The use of ammonia gas is convenient for large-scale operations and can be utilized by bubbling it through the aqueous fermentation medium (fermentation medium) in suitable quantities.

[0267] In the present invention, the pH of the culture medium can be optimized depending on the type of host cell used. For example, the pH range used may be, but is not limited to, 4 to 10, preferably pH 5 to 9, and more preferably pH 5.5 to 7.5.

[0268] In the present invention, the appropriate temperature may be adjusted depending on the type of host cell used. For example, the applicable temperature may be, but is not limited to, 27-45°C, 28-44°C, 29-43°C, 30-42°C, 31-41°C, 32-40°C, or 36-39°C.

[0269] The availability of oxygen and other gases can affect production and fermentation rates. For example, considering oxygen availability, the percentage of dissolved oxygen (DO) in the fermentation medium can range from, but is not limited to, 1-40%, 1.5-35%, 2-30%, 2.5-25%, 3-20%, 4-19%, 5-18%, 6-17%, 7-16%, 8-15%, 9-14%, 10-13%, or 11-12%.

[0270] Fermentation of cells according to the present invention can produce a broth containing the product, a UDCA precursor.

[0271] Methods for purifying products from host cells (i.e., cell lysates) and / or broth are well known in the art. Purification may be based on molecular weight, for example, by size exclusion / exchange chromatography, ultrafiltration through membranes, gel permeation chromatography (e.g., preparative disc-gel electrophoresis), or density centrifugation.

[0272] Additionally, it can be purified based on surface charge or hydrophobicity / hydrophilicity, for example, by isoelectric precipitation, anion / cation exchange chromatography, isoelectric focusing (IEF), or reversed phase chromatography.

[0273] Additionally, based on solubility, it can be purified by, for example, ammonium sulfate precipitation, isoelectric precipitation, surfactant, detergent, or solvent extraction.

[0274] Hereinafter, the present invention will be described in more detail through examples. However, the present invention can be modified in various ways and can take various forms. Therefore, the specific examples and descriptions described below are only intended to aid in understanding the present invention and are not intended to limit the present invention to a specific disclosed form. It should be understood that the scope of the present invention includes all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention.

[0275] [Example 1]

[0276] Construction of an expression plasmid for the conversion of cholesterol to 7α-hydroxycholesterol in yeast.

[0277] To construct a CA synthesis pathway using cholesterol as a substrate in yeast, cholesterol converting enzyme and uptake enzyme were inserted into a yeast expression vector, and step-by-step integrated plasmids were constructed to verify their expression.

[0278] 1-1. Insert gene cloning primers

[0279] All gene units including promoters and terminators were inserted into the pY-sHsCYP7A1 plasmid containing codon-optimized CYP7A1 for yeast derived from H. sapiens. The gene units were inserted sequentially. The first insertion was performed using the primers Con1_NgoMIV_F and Con1_SpeI_r to amplify both ends with the NgoMIV and SpeI restriction sites. The second insertion was performed using the primers Con3_NarI_F and Con3_PacI_r to amplify both ends with the NarI and PacI restriction sites. In the third insertion, the primers Con8_SacII_F and Con8_NarI_r to amplify both ends with the SacII and NarI restriction sites. When making primers, 15-16 bp of homology with the vector at the 5' end was inserted to make them suitable for ligation by SLIC. The above primer information is shown in Table 12.

[0280] Oligonucleotide primers for cloning

[0281] Cloning primer direction sequence size (bp) sequence number Con1_NgoMIV_F forward 5'- gacggggaaagccggc GTCGACCCGCGGGGCGCCTTAATTAA ACGGATTAGAAGCCGCCGAG-3'62 bp91Con1_SpeI_r reverse 5'- tctaatccgtactagt AAAGCCTTCGAGCGTCC-3'33 bp92Con3_NarI_F forward 5'- tcgacccgcggggcgcc ACGGATTAGAAGCCGCCGAG-3'37 bp93Con3_PacI_r reverse 5'- cttctaatccgtttaattaa AGCCTTCGAGCGTCC-3'35 bp94Con8_SacII_F forward 5'- agccggcgtcgacccgcgg ACGGATTAGAAGCCGCCGAG-3'39 bp95Con8_NarI_rReverse 5'- ttctaatccgtggcgcc AAAGCCTTCGAGCGTCC-3'34 bp96

[0282]

[0283] 1-2. Template vector

[0284] A vector, pYES2, measuring approximately 5.8 kb in size, was used to express five genes involved in the conversion of cholesterol to 7α-hydroxycholesterol in yeast. The pYES2 vector is an E. coli-yeast shuttle vector containing the GAL1 promoter and is a yeast expression vector induced by galactose. The five genes were already inserted into the pYES2 vector, and each insert gene was inserted overlappingly into the pYES2 vector, including the promoter and terminator of the pYES2 vector. The base sequence of the template vector is shown in Figure 2.

[0285] 1-3. Insert gene

[0286] Five genes, sHsCYP7A1, sHsPOR, sScNcp1, sHsCYB5, and sScUPC2-1, are codon-optimized genes suitable for yeast expression, and each gene was inserted into the pYES2 vector using SacI / XbaI restriction enzymes for step-by-step integrated plasmid construction. All genes to be constructed were designed to be inserted into the pY-sHsCYP7A1 plasmid into which sHsCYP7A1 was inserted.

[0287] The base sequence of pY-sHsCYP7A1 is as shown in Figure 3a. The base sequences of the four gene expression cassettes to be inserted, excluding the sHsCYP7A1 gene already inserted into the pYES2 vector, are as shown in Figures 3b to 3e.

[0288] 1-4. Plasmid construction

[0289] For the conversion of cholesterol into 7α-hydroxycholesterol in yeast, UPC2-1 from S. cerevisiae, a gene that absorbs cholesterol as a substrate, is involved, and CYP7A1 from H. sapiens, a cytochrome P450 family enzyme, is involved in the conversion of 7α-hydroxycholesterol. For the functional expression of the CYP7A1 enzyme, the POR (Cytochrome P450 oxidoreductase) gene from H. sapiens and the Ncp1 gene from S. cerevisiae were selected as candidate genes for POR (Cytochrome P450 oxidoreductase). In addition, the CYB5 (Cytochrome b5) gene from H. sapiens was selected as cytochrome b5. These genes were used to construct various combinations so that cholesterol can be absorbed into yeast and converted into 7α-hydroxycholesterol, and each expression plasmid was constructed as a unit to verify this. The constructed plasmids were named pY-sCU, pY-sCP, pY-sCN, pY-sCPB, pY-sCPU, pY-sCNB, pY-sCNU, pY-sCPBU, and pY-sCNBU, respectively.

[0290] The construction was performed by using different primers depending on the insertion order. For the first insertion, amplification was performed with Con1_NgoMIV_F and Con1_SpeI_r, treatment with NgoMIV and SpeI, and insertion was performed. For the second insertion, amplification was performed with Con3_NarI_F and Con3_PacI_r, treatment with NarI and PacI, and insertion was performed. For the third insertion, amplification was performed with Con8_SacII_F and Con8_NarI_r, treatment with SacII and NarI, and insertion was performed.

[0291] To confirm the construction results, the suitability of the constructed plasmids was determined through colony PCR and DNA sequencing. The overall construction outline is shown in Figures 4a to 4d. A schematic diagram of the construction of nine expression plasmids is shown in Figure 4e.

[0292] [Example 2]

[0293] Confirmation of conversion of cholesterol to 7α-hydroxycholesterol using yeast

[0294] To construct the 15 steps of the cholesterol to cholic acid (CA) biosynthetic pathway step by step in yeast, the first step was to convert cholesterol as a substrate into 7α-hydroxycholesterol (7HC, CAS No. 566-26-7) in yeast.

[0295] 2-1. Introduction of expression plasmid into yeast

[0296] Saccharomyces cerevisiae strain Y2805 was transformed with 10 stepwise integrated plasmids containing 7α-hydroxycholesterol production and cholesterol uptake genes and 4 plasmids containing truncated forms of the 7α-hydroxycholesterol convertase sHsCYP7A1 gene (14 in total; pY-sHsCYP7A1, pY-sCU, pY-sCP, pY-sCPU, pY-sCPB, pY-sCPBU, pY-sCN, pY-sCNU, pY-sCNB, pY-sCNBU, pY-tsHsCYP7A1, pY-tsCU, pY-tsCPU, pY-tsCPBU) and the empty vector pYES2 as a control. The transformation method was to introduce a total of 15 types of plasmids into yeast strains using the PEG / LiAc method, and colonies were obtained on uracil-deficient plates. In addition to the Y2805 strain, the BY4741, L3262, KCCM 50549, ATCC201228, ATCC201238, ATCC201741, Inv Sc1, and CEN.PK strains were also transformed with expression plasmids using the same method.

[0297] Information on the production strain and plasmid is shown in Tables 13 and 14, respectively.

[0298] Strain Genotype S. cerevisiae Y2805 (hereinafter Y2805) MATa pep4::HIS3 prb1-d can1 GAL2 his3 ura3-52 S. cerevisiae BY4741 (hereinafter BY4741) MATa his3Δ1 leu2Δ met15Δ ura3Δ S. cerevisiae L3262 (hereinafter L3262) MATa ura3-52 leu2-3, 112 his4-34 S. cerevisiae KCCM50549 (hereinafter KCCM50549) MATa / MATα, trp1, his3, leu2, ura3 S. cerevisiaeATCC201228 (hereinafter ATCC201228)MATa ade2-1 ura3 his3 leu2-k::ADE2-URA3::leu2-k hpr1deleta3::HIS3S. cerevisiae ATCC201238 (hereinafter referred to as ATCC201238) MATα leu2-3 leu2-112 trp1-1 ura3-1 his3-11 his3-15 ade2-1 can1-100S. cerevisiae ATCC201741 (hereinafter referred to as ATCC201741) MATα ade2-1 trp1-1 can1-100 leu2-3 leu2-112 his3-11 his3-15 ura3-52 GAL+S. cerevisiaeInv Sc1 (hereinafter Inv Sc1)MATa his3D1 leu2 trp1-289 ura3-52S. cerevisiaeCEN.PK 2-1C (hereinafter CEN.PK)MATa ura3-52 trp 1-289 leu2-3,112 his3-Δ1 MAL2-8C suc2

[0299] Plasmid details pYES2P gal1 E. coliand yeast shuttle vector, 2μ origin, URA3 gene, Amp rpY-sHsCYP7A1pYES2 containing the codon-optimizedCYP7A1gene fromHomo sapienspY-sCUpY-sHsCYP7A1 containing the codon-optimizedUPC2-1gene fromSaccharomyces cerevisiaepY-sCPpY-sHsCYP7A1 containing the codon-optimizedPOR(Cytochrome P450 oxidoreductase)gene fromH. sapienspY-sCPUpY-sCP containing the codon-optimizedUPC2-1gene fromS. cerevisiaepY-sCPBpY-sCP containing the codon-optimizedCYB5gene fromH. sapienspY-sCPBUpY-sCPB containing the codon-optimizedUPC2-1gene fromS. cerevisiaepY-sCNpY-sHsCYP7A1 containing the codon-optimizedNcp1gene fromS. cerevisiaepY-sCNUpY-sCN containing the codon-optimizedUPC2-1gene fromS. cerevisiaepY-sCNBpY-sCN containing the codon-optimizedCYB5(Cytochrome b5)gene fromH. sapienspY-sCNBUpY-sCNB containing the codon-optimizedUPC2-1gene fromS. cerevisiaepY-tsHsCYP7A1pYES2 containing the codon-optimizedCYP7A1gene(truncated form) fromH. sapienspY-tsCUpY-tsHsCYP7A1containing the codon-optimizedUPC2-1gene fromS.cerevisiaepY-tsCPUpY-tsCU containing the codon-optimizedPOR(Cytochrome P450 oxidoreductase)gene fromH. sapienspY-tsCPBUpY-tsCU containing the codon-optimized CYB5(Cytochrome b5)gene fromH. sapiens.

[0300] 2-2. Preparation of media and reagents

[0301] The solid medium for obtaining yeast colonies for seed culture was SD-Ura agar medium lacking uracil, and was prepared and used as follows. 6.7 g of yeast nitrogen base without amino acid and 0.77 g of -Ura DO (drop out) supplement were dissolved in 900 mL of distilled water, and the pH was adjusted to approximately 5.6-6.0. 20 g of agar was added and dissolved. The mixture was autoclaved at 121°C for 20 minutes, cooled, and 100 mL of 20% glucose was added. 20 mL was poured into each petri dish, solidified at room temperature, and then stored in the refrigerator. The seed culture liquid medium was prepared by dissolving 6.7 g of yeast nitrogen base without amino acids and 0.77 g of -Ura DO (drop out) supplement in 900 mL of distilled water, which was the same as the solid medium, and adjusting the pH to 5.6-6.0. The medium was autoclaved at 121°C for 20 minutes, cooled, and then 100 mL of 20% glucose was added. The medium was then stored at room temperature.

[0302] In the case of the expression vector pYES2, since expression is regulated by galactose with the Gal promoter, galactose was added to the yeast culture medium during the main culture to induce expression. For the main culture, a complex medium was used, and when expression was not induced, it was cultured in YPD medium, and when expression was induced, YPDG medium was used. YPD medium was prepared by dissolving 10 g of yeast extract and 20 g of peptone in 900 mL of distilled water, autoclaving at 121℃ for 20 minutes, cooling, and adding 100 mL of 20% glucose. YPDG medium was prepared by dissolving 10 g of yeast extract and 20 g of peptone in 900 mL of distilled water, autoclaving at 121℃ for 20 minutes, cooling, and adding 50 mL of 20% glucose and 50 mL of 20% galactose. After preparation, it was stored at room temperature.

[0303] The addition of cholesterol used as a substrate was performed by making a 5% cholesterol stock in a solution of ethanol and Tween 80 in a 2:1 ratio, adding it to the culture medium at an appropriate concentration, and culturing.

[0304] 2-3. Seed culture and main culture

[0305] The yeast colonies obtained from the plate were inoculated into 50 mL of the prepared seed culture medium and cultured overnight at 30°C and 180 rpm. The cell growth amount was measured using a spectrophotometer at an absorbance of 600 nm, and the measured cell growth amount was added to the main culture medium to obtain an OD of 0.4. 600nm It was calculated and inoculated. After inoculation, it was cultured for up to 120 hours under the conditions of 30℃ and 180 rpm.

[0306] 2-4. Yeast pellet extraction

[0307] To extract cholesterol and 7α-hydroxycholesterol from yeast cells, 50 mL of culture was taken and centrifuged at 3,000 rpm for 30 minutes, the supernatant was separated, and the cell pellet was collected and extracted. 20 mL of resuspension solution (15% KOH (w / v), 0.125% pyrogallol (w / v), 71% MeOH (v / v)) was added to the yeast pellet and vortexed to resuspend the cells. The mixture was heated in a water bath at 85 °C for 2 hours. After cooling, petroleum ether was added to the resuspension solution and vortexed to separate the layers. To ensure good layer separation, centrifugation was performed at 3,000 rpm for 5 minutes and the upper petroleum ether layer was collected. The amount of petroleum ether used was 3 mL each, and this procedure was repeated three times in total. The recovered petroleum ether supernatant was dried and used as a sample for 7α-hydroxycholesterol and cholesterol analysis.

[0308] 2-5. Analysis

[0309] The standard solution was prepared by taking 10 mg of cholesterol and 7α-hydroxycholesterol, diluting them in 10 mL of methanol, and filtering them through a 0.45 μm filter. The test solution was prepared by diluting the concentrated and dried sample with 2 mL of methanol, and then filtering them through a 0.45 μm filter.

[0310] The production of cholesterol and 7α-hydroxycholesterol was confirmed using the peak area of ​​the test solution obtained by testing the blank solution (methanol), standard solution, and test solution under the operating conditions of Table 15. The gradient mode is as shown in Table 16.

[0311] System Separation Module, Charged Aerosol Detector Column Waters Symmetry C18 (4.6 x 75 mm, 3.5 μm) Column Temperature 20 °C Detector Temperature 30 °C Detector Collection Rate 20 Hz Detector Filter 5.0 s Detector Gas Mode Analysis Detector Power Function 1 Flow Rate 1.2 mL / min Mobile Phase A 100% MeOH Mobile Phase B 100% ACN Injection Volume 10 μL Sample Temperature 4 °C Autosampler Extraction Rate 5.0 μL / s Run Time 45 min Needle Wash 80% Methanol Seal Wash 10% Methanol

[0312] Time (min)Flow (ml / min)A (%)B (%)0.00.3208018.00.3208018.11.2802040.00.3208045.00.32080

[0313] 2-6. Confirmation of 7α-hydroxycholesterol production by 14 types of 7α-hydroxycholesterol-producing plasmids

[0314] Among the 14 candidate plasmids for 7α-hydroxycholesterol production, the production plasmid was primarily selected by confirming whether it produced 7α-hydroxycholesterol. YPD and YPDG media were used for 72 hours of culture, depending on the presence or absence of galactose induction. In the case of the empty vector pYES2, it was cultured in the presence or absence of cholesterol to determine whether the yeast itself produced cholesterol, and for the remaining 14 types, all were cultured with cholesterol added to the culture medium. The cell growth amount as a result of the main culture is as shown in Figure 5a, and the results of the 7α-hydroxycholesterol and cholesterol analysis are as shown in Table 17.

[0315] No.균주배양배지ppm(mg / L)7α-하이드록시콜레스테롤콜레스테롤1S. cerevisiaeY2805 pYES2 (w / o cholesterol)YPDN.DN.DYPDGN.DN.D2S. cerevisiaeY2805 pYES2 (w / cholesterol)YPDN.D3.7YPDGN.D3.83S. cerevisiaeY2805 pY-sHsCYP7A1YPDN.D4.7YPDGN.D2.54S. cerevisiaeY2805 pY-sCUYPDN.D6.8YPDGDetected6.95S. cerevisiaeY2805 pY-sCPYPDN.D6.1YPDGN.D4.56S. cerevisiaeY2805 pY-sCPUYPDN.D3.9YPDGDetected5.67S. cerevisiaeY2805 pY-sCPBYPDN.D6.0YPDGN.D5.18S. cerevisiaeY2805 pY-sCPBUYPDN.D6.9YPDGDetected15.69S. cerevisiaeY2805 pY-sCNYPDN.D6.3YPDGDetected6.910S. cerevisiaeY2805 pY-sCNUYPDN.D11.2YPDGN.D6.511S. cerevisiaeY2805 pY-sCNBYPDN.D6.1YPDGN.D9.412S. cerevisiaeY2805 pY-sCNBUYPDN.D6.5YPDG0.316.713S. cerevisiaeY2805 pY-tsHsCYP7A1YPDN.D7.7YPDGDetected13.914S. cerevisiaeY2805 pY-tsCUYPDN.D8.0YPDGDetected8.215S. cerevisiaeY2805 pY-tsCPUYPDN.D7.1YPDGN.D9.316S. cerevisiaeY2805 pY-tsCPBUYPDN.D4.1YPDGN.D7.8

[0316] *ND: Not detected

[0317] When examining whether 7α-hydroxycholesterol and cholesterol were produced within cells depending on the presence or absence of cholesterol addition, it was confirmed that neither 7α-hydroxycholesterol nor cholesterol was produced within the cells by the yeast itself when cholesterol was not added. Only when cholesterol was exogenously added during culture, was cholesterol taken up into the cells and cholesterol was confirmed within the cells. In addition, no sample showed 7α-hydroxycholesterol production in YPD medium without galactose, an inducer of the Gal promoter. Based on these results, only YPDG was used in future experiments. Among the 14 constructed 7α-hydroxycholesterol-producing plasmids, 7α-hydroxycholesterol production was confirmed in 7 plasmids: pY-sCU, pY-sCPU, pY-sCPBU, pY-sCN, pY-sCNBU, pY-tsHsCYP7A1, and pY-tsCU.

[0318] 2-7. Selection of 7α-hydroxycholesterol production plasmid

[0319] Among the 14 candidate plasmids for 7α-hydroxycholesterol production, a reproducibility test was performed using 7 plasmids (pY-sCU, pY-sCPU, pY-sCPBU, pY-sCN, pY-sCNBU, pY-tsHsCYP7A1, and pY-tsCU) that were confirmed to produce 7α-hydroxycholesterol. YPDG medium inducing galactose was used for 96 hours, and all samples were constructed in duplicate per condition. In addition, pYES2 plasmid was also cultured as a negative control under the same culture conditions. The cell growth amount as a result of the main culture is as shown in Figure 5b, and the results of 7α-hydroxycholesterol and cholesterol analysis are as shown in Table 18.

[0320] No. Strain ppm (mg / L) 7α-Hydroxycholesterol Cholesterol 1S. cerevisiaeY2805pYES2N.D2.72S. cerevisiaeY2805 pY-sCU0.39.53S. cerevisiaeY2805 pY-sCPUDetected9.94S. cerevisiaeY2805pY-sCPBU0.87.05S. cerevisiaeY2805 pY-sCNDetectedDetected6S. cerevisiaeY2805pY-sCNBU0.36.87S. cerevisiaeY2805pY-tsHsCYP7A1N.D3.58S. cerevisiaeY2805 pY-tsCUN.D5.9

[0321] The reproducibility test results showed that among the seven 7α-hydroxycholesterol-producing plasmids, the pY-sCPBU plasmid had the highest average 7α-hydroxycholesterol productivity. Therefore, pY-sCPBU was selected as the 7α-hydroxycholesterol-producing plasmid and subsequent experiments were conducted.

[0322] 2-8. Selection of yeast strains producing 7α-hydroxycholesterol

[0323] A yeast strain selection test was conducted using the selected pY-sCPBU plasmid. In addition to the Y2805 strain, eight yeast strains were additionally used in the 7α-hydroxycholesterol production test: BY4741, L3262, KCCM 50549, ATCC201228, ATCC201238, ATCC201741, Inv Sc1, and CEN.PK.

[0324] The final culture time was 96 hours, and all samples were constructed in duplicate for each condition. The pYES2 plasmid was also cultured as a negative control under the same culture conditions to determine whether 7α-hydroxycholesterol was productive in various strains when the 7α-hydroxycholesterol production plasmid was not expressed. The cell growth amount for the final 96 hours of the main culture is as shown in Table 19, and the results of the 7α-hydroxycholesterol and cholesterol analysis are as shown in Table 20.

[0325] No. Strain plasmid OD 600nm 1S. cerevisiaeY2805pYES250.42pY-sCPBU36.13S. cerevisiaeBY4741pYES243.14pY-sCPBU45.65S. cerevisiaeL3262pYES253.16pY-sCPBU39.87S. cerevisiaeKCCM 50549pYES216.88pY-sCPBU16.39S. cerevisiaeATCC201228pYES221.010pY-sCPBU23.811S. cerevisiaeATCC201238pYES222.512pY-sCPBU21.313S. cerevisiaeATCC201741pYES222.514pY-sCPBU21.015S. cerevisiaeInvSc1pYES245.016pY-sCPBU40.817S. cerevisiaeCEN.PKpYES254.218pY-sCPBU46.6

[0326] No. Strain Plasmid ppm (mg / L) 7α-Hydroxycholesterol Cholesterol 1 S. cerevisiae Y2805 pYES2N. D5. 32 pY-sCPBU 1. 5 14. 23 S. cerevisiae BY4741 pYES2N. D7. 14 pY-sCPBU 3. 0 13. 95 S. cerevisiae L3262 pYES2N. D6. 06 pY-sCPBUN. D6. 67 S. cerevisiae KCCM 50549 pYES2N. D4. 18 pY-sCPBUN. D9. 29 S. cerevisiae ATCC 201228 pYES2N. D8. 210 pY-sCPBUN. D8. 7 11 S. cerevisiaeATCC201238pYES2N.D8.212pY-sCPBU0.613.313S. cerevisiaeATCC201741pYES2N.D7.814pY-sCPBU0.34.415S. cerevisiaeInvSc1pYES2N.D11.016pY-sCPBU0.19.617S. cerevisiaeCEN.PKpYES2N.D5.918pY-sCPBU0.811.1

[0327] As a result of the production test, 7α-hydroxycholesterol production was confirmed in the existing Y2805 strain and the new BY4741, ATCC201238, ATCC201741, InvSc1, and CEN.PK. Among the five yeast strains confirmed to produce 7α-hydroxycholesterol, BY4741 was confirmed to have the highest 7α-hydroxycholesterol production, and thus was selected as the 7α-hydroxycholesterol-producing yeast strain for subsequent experiments.

[0328] 2-9. Production of 7α-hydroxycholesterol according to the concentration of added cholesterol

[0329] The optimal substrate concentration was determined using BY4741 and the 7α-hydroxycholesterol-producing plasmid pY-sCPBU. The existing substrate concentration was 1000 mg / L, and the amount of substrate absorbed or converted into cells was confirmed to be less than 20 mg / L. Therefore, 7α-hydroxycholesterol production tests were performed by adding substrate at concentrations below 1000 mg / L, without addition, and at levels of 100 mg / L and 500 mg / L.

[0330] All samples were performed in duplicate for each condition, and the cell growth amount as a result of the main culture is as shown in Figure 6a, and the results of the 7α-hydroxycholesterol and cholesterol analysis are as shown in Table 21.

[0331] No. Strain Substance Concentration ppm (mg / L) 7α-Hydroxycholesterol Cholesterol 1 S. cerevisiae BY4741 (pY-sCPBU) Cholesterol 0 mg / L N.DN.D 2 Cholesterol 100 mg / L 2.6 2.63 Cholesterol 500 mg / L 1.5 3.44 Cholesterol 1000 mg / L 0.6 4.1

[0332] *ND: Not detected

[0333] As a result of the production test, when no cholesterol was added, the production of cholesterol and 7α-hydroxycholesterol was not confirmed, and only when cholesterol was added, the production of cholesterol and 7α-hydroxycholesterol within the cells was confirmed. As a result of this experiment, it was confirmed that the overall 7α-hydroxycholesterol production was lower than the previous experiment results even when the existing 1000 mg / L substrate was added. It is thought that this is because the concentration of the substrate absorbed within the cells is relatively low, resulting in low 7α-hydroxycholesterol productivity. However, although the production of 7α-hydroxycholesterol was relatively low, the production of 7α-hydroxycholesterol according to the amount of substrate added was confirmed to be the highest when 100 mg / L was added.

[0334] 2-10. 7α-Hydroxycholesterol production by culture time

[0335] In the case of the existing main culture, it was conducted for 96 hours, but in order to confirm the amount of 7α-hydroxycholesterol production by culture time period, cell growth rate and 7α-hydroxycholesterol productivity were checked at 24-hour intervals for a total culture time of 120 hours. Cholesterol, as a substrate, was added at 1000 mg / L, and all samples were composed of two per condition (duplicate) and performed. In addition, the pYES2 plasmid was also cultured as a negative control under the same culture conditions to confirm the cell growth rate pattern with the 7α-hydroxycholesterol-producing strain. As a result of the main culture, the amount of cell growth is as shown in Figure 6b, and the results of the 7α-hydroxycholesterol and cholesterol analysis are as shown in Table 22.

[0336] No. Strain Culture time (hours) ppm (mg / L) 7α-hydroxycholesterol Cholesterol 1 S. cerevisiae BY4741 (pYES2) 24 N.D2. 42 48 N.D4. 13 72 N.D4. 64 96 N.D5. 55 120 N.D6. 26 S. cerevisiae BY4741 (pY-sCPBU) 240.63.37 482.88.487 23.011.299 62.611.910 1202.918.9

[0337] *ND: Not detected

[0338] In terms of cell growth rate, both the empty vector and the 7α-hydroxycholesterol production plasmid were confirmed to stop growing after 72 hours of culture. 7α-hydroxycholesterol productivity also showed maximum productivity after 72 hours of culture, confirming that the conversion from cholesterol to substrate was already complete. However, the amount of cholesterol absorbed into the cells was confirmed to increase up to 120 hours of culture. Therefore, the results of this experiment confirmed that the culture time for 7α-hydroxycholesterol production is more than 72 hours.

[0339] Item selection conditions Yeast strain S. cerevisiae BY4741 Production plasmid pY-sCPBU Production medium YPDG Substrate addition concentration Cholesterol 100 mg / L Culture time Cultivation 72 hours (30°C, 180 rpm)

[0340] A 7α-hydroxycholesterol production test was conducted using a total of 14 candidate plasmids for 7α-hydroxycholesterol production, and the optimal combination was found to be pY-sCPBU, which is composed of CYP7A1, a gene that converts cholesterol to 7α-hydroxycholesterol, POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5) genes, which are redox partner enzymes of CYP7A1, and UPC2-1 gene, a cholesterol absorption gene. In addition, the redox partner gene of human (H. sapiens) origin, which is of the same origin as the conversion gene, showed the highest 7α-hydroxycholesterol productivity. And it was confirmed that the full-length CYP7A1 gene is more suitable for producing 7α-hydroxycholesterol in yeast than the truncated form, and in the case of the strain, the BY4741 yeast strain had high 7α-hydroxycholesterol productivity. The main function of cytochrome P450 enzymes is the monooxygenation reaction for various substrates, which requires molecular oxygen (O2) and reducing substances of NADPH. These are largely divided into two types, Class I and Class II, depending on the function and component. Class I is a mitochondrial enzyme, and flavin enzyme, ferredoxin reductase, iron sulfur enzyme, and ferredoxin are involved in electron transfer, and NADH or NADPH transfers electrons to the terminal P450 enzyme. Class II, on the other hand, is a microsomal enzyme, and electron transfer is directly from NADPH to the P450 enzyme by catalysis of a flavin enzyme, an NADPH-cytochrome P450 reductase, each containing one molecule of FAD and one molecule of FMN.Some P450 species receive a second electron from another electron transport chain, which consists of NADH-cytochrome b5 reductase and cytochrome b5 in microsomes.

[0341] Experimental results showed that CYP7A1, a 7α-hydroxycholesterol convertase, was expressed more efficiently when NADPH-cytochrome P450 reductase and cytochrome b5 were simultaneously expressed. Based on these results, we were able to identify a gene combination for the expression of CYP7A1, a cytochrome P450 known to be poorly expressed in microorganisms, and it is believed that this can be applied to the expression of cytochrome P450 genes of the same class as CYP7A1 within the cholesterol-CA production pathway.

[0342] [Example 3]

[0343] Construction of a novel 7α-hydroxycholesterol-producing plasmid from mammalian genetic resources

[0344] We sought to construct a novel plasmid that produces 7α-hydroxycholesterol from cholesterol by exploring genetic resources derived from various mammals.

[0345] The novel 7α-hydroxycholesterol production plasmid contains the same cholesterol absorption gene, UPC2-1, as the existing plasmid, pY-sCPBU, but the three major genes that convert 7α-hydroxycholesterol (CYP7A1, POR (Cytochrome P450 oxidoreductase), CYB5 (Cytochrome b5)) were synthesized from genes derived from four new mammalian species (Oryctolagus cuniculus, Gallus gallus, Bos Taurus, and Sus scrofa) rather than the existing human (H. sapiens)-derived ones, thereby constructing the plasmid.

[0346] 3-1. Template vector

[0347] For the production of 7α-hydroxycholesterol, the pYES2 vector (Figs. 6a and 6b) was used as a yeast expression vector, and the genes in the vector were inserted using the SacI and XbaI restriction enzyme sites in the multi-cloning site of the pYES2 vector.

[0348] 3-2. Insert gene

[0349] Three major genes that convert 7α-hydroxycholesterol, CYP7A1, POR (Cytochrome P450 oxidoreductase), and CYB5 (Cytochrome b5), were selected from O. cuniculus, G. gallus, B. Taurus, and S. scrofa, respectively, and codon-optimized for yeast to synthesize a total of 12 genes. The synthesized genes were obtained in the form of insertions into the pYES2 vector using SacI and XbaI restriction enzyme sites, and the nucleotide sequences of each expression cassette are as shown in Figures 7a to 7l.

[0350] 3-3. Cloning primer

[0351] To construct 12 types of plasmids, the expression cassettes containing each gene were amplified using the primers in Table 24 to construct the target plasmids.

[0352] Cloning primer direction sequence size (bp) sequence number POR_NgoMIV_F forward 5'- gacggggaaagccggcgtcgacccgcggggcgccttaattaaacggattagaagccgccgag -3'62 bp 97 POR_SpeI_r reverse 5'- ctaatccgtggactagtaaagccttcgagcgtccc -3'35 bp 98 CYB5_NarI_F forward 5'- tcgacccgcggggcgcc acggattagaagccgccgag -3'38 bp 99 CYB5_PacI_r reverse 5'- cttctaatccgtttaattaaagccttcgagcgtccc -3'36 bp 100 UPC2-1_SacII_F forward 5'- ccggcgtcgacccgcggacggattagaagccgccgag -3'37 bp101UPC2-1_NarI_rReverse5'- ttctaatccgtggcgccaaagccttcgagcgtccc -3'35 bp102

[0353] 3-4. Construction of a novel 7α-hydroxycholesterol-producing plasmid

[0354] During construction, the order was as follows: POR (Cytochrome P450 oxidoreductase), CYB5 (Cytochrome b5), and UPC2-1 gene expression cassettes were inserted into the pY-sCYP7A1 plasmid containing the CYP7A1 gene.

[0355] First, the schematic diagram of the POR (Cytochrome P450 oxidoreductase) gene expression cassette insertion is as shown in Fig. 8a, and the pY-sPOR plasmid (pY-sOcPOR, pY-sGgPOR, pY-sBtPOR, pY-sSsPOR) was used as a template and PCR amplified with the primers POR_NgoMIV_F and POR_SpeI_r. The amplified PCR product was treated with the restriction enzyme sites NgoMIV and SpeI, and the POR (Cytochrome P450 oxidoreductase) gene-derived product was inserted into each of the pY-sOcCYP7A1, pY-sGgCYP7A1, pY-sBtCYP7A1, and pY-sSsCYP7A1 plasmids treated with the same restriction enzyme sites. As a result, four types of pY-sOcCP, pY-sGgCP, pY-sBtCP, and pY-sSsCP plasmids were constructed.

[0356] The CYB5 (Cytochrome b5) gene was inserted into the pY-sOcCP, pY-sGgCP, pY-sBtCP, and pY-sSsCP plasmids constructed above, and the expression cassette insertion schematic is as shown in Fig. 8b. For construction, pY-sOcCYB5, pY-sGgCYB5, pY-sBtCYB5, and pY-sSsCYB5 were used as templates and PCR amplified with the primers CYB5_NarI_F and CYB5_PacI_r. The amplified PCR products were treated with the restriction enzyme sites NarI and PacI, and the CYB5 (Cytochrome b5) gene derivatives were inserted into each of the pY-sOcCP, pY-sGgCP, pY-sBtCP, and pY-sSsCP plasmids treated with the same restriction enzyme sites. As a result, four types of pY-sOcCPB, pY-sGgCPB, pY-sBtCPB, and pY-sSsCPB plasmids were constructed.

[0357] Finally, the UPC2-1 gene was inserted into the pY-sOcCPB, pY-sGgCPB, pY-sBtCPB, and pY-sSsCPB plasmids constructed above, and the expression cassette insertion schematic is as shown in Fig. 8c. For construction, the pY-sScUPC2-1 plasmid was used as a template and PCR amplified with the primers UPC2-1_SacII_F and UPC2-1_NarI_r. The amplified PCR product was treated with the restriction enzyme sites SacII and NarI, and the UPC2-1 gene expression cassette was inserted into the pY-sOcCPB, pY-sGgCPB, pY-sBtCPB, and pY-sSsCPB plasmids treated with the same restriction enzyme sites. As a result, four types of plasmids, pY-sOcCPBU, pY-sGgCPBU, pY-sBtCPBU, and pY-sSsCPBU, were constructed. The suitability of the constructed plasmids was determined through plasmid DNA fragmentation.

[0358] The results of DNA fragmentation of the plasmids pY-sOcCPBU, pY-sGgCPBU, pY-sBtCPBU, and pY-sSsCPBU, which were finally constructed among the 12 types, using restriction enzymes SacII, PacI, and SpeI are as shown in Figure 9. The expected fragment sizes when cut using the three restriction enzymes are 7,171 bp, 4,604 bp, and 2,821 bp for pY-sOcCPBU, 7,207 bp, 4,724 bp, and 2,812 bp for pY-sGgCPBU, 7,168 bp, 4,712 bp, and 2,824 bp for pY-sBtCPBU, and 7,171 bp, 4,712 bp, and 2,818 bp for pY-sSsCPBU. The experimental results confirmed that it was suitable for the size.

[0359] [Example 4]

[0360] Production of 7α-hydroxycholesterol from various genetic sources using yeast.

[0361] In order to produce 7α-hydroxycholesterol, the first step of the 15 steps of the CA production pathway from cholesterol in yeast, we secured transgenes from four new mammalian species in addition to the existing transgene from human (Homo sapiens) to confirm 7α-hydroxycholesterol production and select new genetic resources with improved productivity.

[0362] 4-1. Introduction of expression plasmid into yeast

[0363] To improve 7α-hydroxycholesterol productivity, three major genes involved in the conversion of cholesterol to 7α-hydroxycholesterol (CYP7A1, POR (Cytochrome P450 oxidoreductase), CYB5 (Cytochrome b5)) were obtained from four new strains of origin and their effects on 7α-hydroxycholesterol productivity were investigated.

[0364] Saccharomyces cerevisiae BY4741 was transformed with five 7α-hydroxycholesterol producing plasmids, pY-sCPBU, pY-sOcCPBU, pY-sGgCPBU, pY-sBtCPBU, and pY-sSsCPBU, respectively. The transformation was performed using a yeast transformation kit (Sc EasyComp TM Transformation Kit) was used, and the transformation method was carried out according to the protocol (Version C, 110801) included in the kit. The method is largely divided into the production of competent cells for transformation and transformation. Competent cells were produced by washing the cells with solution I in the kit using cells in the exponential growth phase (mid-log phase) and resuspending them with solution II. Transformation was performed by adding solution III to the mixture of prepared competent cells and plasmid and reacting in a 30°C water bath for 1 hour.

[0365] The medium and reagents were prepared and used in the same manner as in Example 1-2, and the culture was conducted in the same manner as in 1-3. At this time, cholesterol was added to the culture medium so that the final concentration was 100 mg / L.

[0366] 4-2. Extraction

[0367] Extraction was performed using two methods. To confirm the production of cholesterol and 7α-hydroxycholesterol, a cholesterol purification method was used, and to confirm the productivity of the second-stage product, 7α-hydroxy-4-cholesten-3-one (7α-Hydroxy-4-cholesten-3-one, CAS No. 3862-25-7), extraction was performed using an extraction method named M8 method below from yeast.

[0368] Cholesterol extraction was performed as follows. After completion of culture, the cell pellet collected from 50 mL of culture medium was resuspended in 20 mL of a resuspension solution (15% KOH (w / v), 0.125% pyrogallol (w / v), 71% MeOH (v / v)), and the mixture was reacted at 85°C to cause cell disruption and saponification. After the reaction, extraction was performed with petroleum ether, and the extraction solvent was dried to prepare an analysis sample.

[0369] The M8 method was performed as follows. After completion of culture, 20 mL of 50 mM Tris-Cl (pH 7.5) and approximately 1.0–1.5 g of glass beads were added to the cell pellet recovered from 50 mL of culture medium. The cells were disrupted by resuspension and vortexing. The pellet was extracted three times with ethyl acetate, and the extraction solvent was dried to prepare an analytical sample.

[0370] 4-3. Analysis

[0371] The standard solution was prepared by taking 10 mg of cholesterol, 7α-hydroxycholesterol, and 7α-hydroxy-4-cholesten-3-one, diluting them in 10 mL of methanol, and then filtering them through a 0.22 μm filter. The test solution was prepared by diluting the concentrated and dried sample with 2 mL of methanol, and then filtering them through a 0.22 μm filter. The production of cholesterol and 7α-hydroxycholesterol was confirmed using the peak areas of the test solution obtained using the two operating conditions of Table 25 or Table 15 using LC-CAD with the blank test solution (methanol), standard solution, and test solution. Each gradient mode is as shown in Tables 26 and 16.

[0372] System HPLC-CAD Column Waters symmetry C18 (4.6 x 75 mm, 3.5 μm) Column temperature 20 °C Detector temperature 30 °C Detector collection rate 20 Hz Detector filter 5.0 s Detector gas mode Analysis Detector power Function 1 Mobile phase A Methanol : Water : Formic acid = 35 : 65 : 0.65 Mobile phase B Methanol : Acetonitrile : Water : Formic acid = 50 : 47 : 3 : 0.65 Injection volume 1 μL Sample temperature 4 °C Automatic sampler Extraction rate 5.0 μL / s Run time 35 min Needle wash 80% methanol Seal wash 10% methanol

[0373] Time (min)Flow (ml / min)A (%)B (%)0.00.3406010.00.3109025.00.3109030.00.3406030.10.3406035.00.34060

[0374] The production of 7α-hydroxy-4-cholesten-3-one was confirmed using the peak area of ​​the test solution obtained by testing the blank test solution (methanol), standard solution, and test solution under the operating conditions of Table 27 in LC-MS.

[0375] LC System Column Waters symmetry C18 (4.6 x 75 mm, 3.5 μm) Column Temperature 25°C Flow Rate 1.2 mL / min Mobile Phase A 100% MeOH Mobile Phase B 100% ACN Injection Volume 10 μL Sample Temperature 4°C Autosampler Extraction Rate 5.0 μL / s Run Time 45 min Needle Wash 80% Methanol Seal Wash 10% Methanol Gradient Mode Time (min) Flow (ml / min) A (%) B (%) 0 0.6 2 0 8 0 8 0.6 2 0 8 0 8 5 1 0 8 0 2 0 1 6 5 1 0 8 0 2 0 1 7 1 0 2 0 8 0 2 0 1 0 2 0 8 0 MS System Ionization Source APCI Corona 4.0 μA Cone 25 V APCI Probe Temperature 350°C Gas Desolvation Flow 400 L / hr RM Resolution 12.9HM Resolution 115Ion Energy 10.8RM Resolution 22.8HM Resolution 215Ion Energy 20.5MRM Mode Compound Parent Ion Daughter Ion Cholesterol 396.3147.07α-Hydroxycholesterol 367.5241.07α-Hydroxy-4-cholesten-3-one 401.3383.5

[0376] 4-4. Primary cultivation for 7α-hydroxycholesterol production using various genetic resources

[0377] After transformation of yeast with the empty vector pYES2, the existing 7α-hydroxycholesterol production plasmid pY-sCPBU, and 7α-hydroxycholesterol production plasmids constructed from four new species (pY-sOcCPBU, pY-sGgCPBU, pY-sBtCPBU, pY-sSsCPBU), the production of 7α-hydroxycholesterol was confirmed by culturing.

[0378] As a result of the culture, as confirmed in Figure 10a, in the case of the main culture cell growth, the OD of the empty vector pYES2 after the final culture 600nm While the OD of the five 7α-hydroxycholesterol producing plasmids was approximately 52 600nmIt was confirmed that the cell growth rate was generally lower than that of the empty vector, at about 42. As a result of checking the productivity of 7α-hydroxycholesterol, a trace amount of cholesterol was confirmed to be absorbed into the cells in the empty vector pYES2, but 7α-hydroxycholesterol production could not be confirmed. In the case of the existing 7α-hydroxycholesterol-producing plasmid, pY-sCPBU, it was confirmed that approximately 1.9 mg / L of 7α-hydroxycholesterol was produced, and among the new plasmids, pY-sOcCPBU derived from O. cuniculus was confirmed to have the highest productivity, producing approximately 2.0 mg / L of 7α-hydroxycholesterol. As shown in Table 28, three of the four new plasmids (O. cuniculus, B. taurus, S. scrofa) produced 7α-hydroxycholesterol, and G. pY-sGgCPBU derived from gallus showed intracellular cholesterol uptake, but conversion to 7α-hydroxycholesterol was not confirmed.

[0379] No. Strain Plasmid Concentration (mg / L) 7α-Hydroxycholesterol Cholesterol 1BY474 1pYES2N.D. 0.72pY-sCPBU 1.94.13pY-sOcCPBU 2.05.24pY-sGgCPBUN.D. 6.95pY-sBtCPBU 0.65.46pY-sSsCPBU 0.43.0

[0380] * ND: Not detected

[0381] 4-5. Secondary culture to confirm the reproducibility of 7α-hydroxycholesterol production

[0382] To confirm reproducibility, the same test was performed repeatedly. As shown in Fig. 10b, the cell growth rate was generally lower compared to the primary culture, but the cell growth rate of the 7α-hydroxycholesterol production plasmid was lower than that of the empty vector pYES2. As shown in Table 29, in the case of 7α-hydroxycholesterol production, no production was observed when using the empty vector and pY-sGgCPBU plasmid, as in the primary culture. In the case of pY-sOcCPBU, the primary culture produced approximately 2.0 mg / L of 7α-hydroxycholesterol, but the productivity was slightly reduced to approximately 1.4 mg / L in the secondary culture, and the existing 7α-hydroxycholesterol plasmid, pY-sCPBU, was confirmed to produce approximately 2.0 mg / L of 7α-hydroxycholesterol, showing production at a level equivalent to that of the primary culture. pY-sBtCPBU and pY-sSsCPBU were confirmed to produce at a level similar to that of the primary culture in the reproduction test as well. Therefore, the results of this reproduction test confirmed that the existing pY-sCPBU and pY-sOcCPBU have high 7α-hydroxycholesterol production.

[0383] No. Strain Plasmid Concentration (mg / L) 7α-Hydroxycholesterol Cholesterol 1BY4741pYES2N.D.0.82pY-sCPBU2.08.43pY-sOcCPBU1.48.94pY-sGgCPBUN.D.9.15pY-sBtCPBU0.58.86pY-sSsCPBU0.22.4

[0384] * ND: Not detected

[0385] 4-6. Third culture for comparison of the two-step material production of the CA pathway

[0386] In the existing results, it was confirmed that 7α-hydroxycholesterol produced by a nonspecific reaction by genes in yeast during the production of the first step material was also converted to 7α-hydroxy-4-cholesten-3-one, a second step product in the CA pathway. Therefore, based on the above results, two types of plasmids (pY-sCPBU, pY-sOcCPBU) were selected for having the highest 7α-hydroxycholesterol production, and the productivity of 7α-hydroxycholesterol and 7α-hydroxy-4-cholesten-3-one were evaluated to select the final 7α-hydroxycholesterol-producing plasmid. For this test, the same conditions as the first and second cultures were used, but the same sample was extracted using the cholesterol purification method for the extraction method to confirm 7α-hydroxycholesterol production and the M8 extraction method for the extraction method to confirm 7α-hydroxy-4-cholesten-3-one production, and then the productivity was confirmed.

[0387] As a result of the culture, as shown in Fig. 10c, the cell growth amount and 7α-hydroxycholesterol production in the third culture were confirmed at the same level as in the first and second cultures. In addition, as a result of confirming the productivity of 7α-hydroxy-4-cholesten-3-one, as shown in Table 30, pY-sOcCPBU produced approximately 6.0 ㎍ / L, whereas the existing pY-sCPBU plasmid produced approximately 7.8 ㎍ / L, confirming that the existing plasmid had a higher productivity of 7α-hydroxy-4-cholesten-3-one, although the amount was small. Therefore, based on the test results up to the third culture, the optimal combination among the novel mammalian one-step conversion genes is pY-sOcCPBU derived from O. cuniculus, but the existing 7α-hydroxycholesterol producing plasmid, H. Compared to pY-sCPBU derived from S. sapiens, it was confirmed that the productivity of 7α-hydroxycholesterol and 7α-hydroxy-4-cholesten-3-one was low.

[0388] No. Strain Plasmid Concentration mg / L (ppm) ㎍ / L (ppb) 7α-Hydroxycholesterol Cholesterol 2 Step 1 BY4741 pYES2 N.D. 1.4 ND2 pY-sCPBU 1.9 6.9 7.8 3 pY-sOcCPBU 1.5 8.6 6.0

[0389] * ND: Not detected, Step 2: 7α-hydroxy-4-cholesten-3-one

[0390] [Example 5]

[0391] Construction of a plasmid producing 7α-hydroxy-4-cholesten-3-one using the ERG26 gene and confirmation of 7α-hydroxy-4-cholesten-3-one production.

[0392] By inserting the yeast-derived ERG26 gene (sterol-4alpha-carboxylate 3-dehydrogenase (decarboxylating), 1.1.1.170) into a plasmid for producing 7α-hydroxycholesterol, a plasmid capable of producing 7α-hydroxy-4-cholesten-3-one, a step 2 substance in the CA biosynthetic pathway from cholesterol, was constructed. Using this, we attempted to determine whether the productivity of 7α-hydroxy-4-cholesten-3-one, a step 2 substance, and 4-cholesten-7α,12α-diol-3-one, a step 3 substance, from cholesterol in yeast could be improved.

[0393] 5-1. Template vector

[0394] The 7α-hydroxycholesterol production plasmid for ERG26 gene insertion used was pY-sCPBU.

[0395] 5-2. Insert gene

[0396] The ERG26 gene, synthesized by codon optimization in yeast derived from Saccharomyces cerevisiae, was used in the form of the pY-sScERG26 plasmid inserted into the pYES2 vector using the SacI and XbaI restriction enzyme sites. The nucleotide sequence of the corresponding expression cassette is as shown in Fig. 11.

[0397] 5-3. Cloning primer

[0398] Using the primers in Table 31, the pY-sScERG26 plasmid containing the ERG26 gene was amplified as a template and used as an insertion product.

[0399] Cloning primer direction Sequence size (bp) Sequence number sScERG26_SalI_F Forward 5'- gaaagccggcgtcgacacggattagaagccgccgag -3'36 bp 103 sScERG26_SacII_r Reverse 5'- gcttctaatccgtccgcggaaagccttcgagcgtccc -3'37 bp 104

[0400] 5-4. Construction of a plasmid producing 7α-hydroxy-4-cholesten-3-one

[0401] A schematic diagram of the ERG26 gene expression cassette insertion is shown in Figure 12. Using the pY-sScERG26 plasmid as a template, sScERG26_SalI_F and sScERG26_SacII_r were used as primers for PCR amplification. The amplified PCR product was treated with restriction enzyme sites SalI and SacII, and the treated PCR product was inserted into the pY-sCPBU plasmid treated with the same restriction enzyme sites.

[0402] The suitability of the constructed plasmid was determined through plasmid DNA fragmentation. After completion of construction, the results of DNA fragmentation of the pY-sCPBUE plasmid using the restriction enzymes NheI and XhoI are shown in Figure 13. The expected fragment sizes when cut using the two restriction enzymes are 14,707 bp and 1,836 bp, respectively, and the experimental results confirmed that the fragments were suitable for the corresponding sizes.

[0403] 5-5. Production strains and plasmids

[0404] The production strain is Saccharomyces cerevisiae BY4741, and the plasmid information is as shown in Table 32.

[0405] Plasmid detailed description reference pYES2P gal1 E. coliand yeast shuttle vector, 2μ origin, URA3 gene, Amp r empty vector pYES_HISP gal1 E. coliand yeast shuttle vector, 2μ origin, HIS3 gene, Amp r Empty vector pY-sCPBUpYES2 containing the codon-optimized CYP7A1, POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5) genes from Homo sapiens and UPC2-1 gene from Saccharomyces cerevisiaeCA pathway step 1 material production plasmid pY-sCPBUDpY-sCPBU containing the codon-optimized HDS3B7 gene from H. sapiensCA pathway step 2 material production plasmid pY-sCPBUDCpY-sCPBUD containing the codon-optimized CYP8B1 gene from H. sapiensCA pathway step 3 material production plasmid pY-sCPBUEpY-sCPBU containing the codon-optimized ERG26 gene from S. Plasmid for producing the second step of the CA pathway, pY-sCPBUECpY-sCPBUE containing the codon-optimized CYP8B1 gene from H. sapiens. Plasmid for producing the third step of the CA pathway, pYH-sHBpYES_HIS containing the codon-optimized POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5) genes from H. sapiens. Plasmid containing the redox partner enzymes for producing the third step of the CA pathway.

[0406] 5-6. Introduction of expression plasmid into yeast

[0407] To introduce the newly constructed plasmid into yeast, Invitrogen's Sc EasyComp TM A transformation kit was used. The transformation method was carried out according to the protocol in the kit (Version C, 110801), and the transformation was performed in the same manner as in Example 3-1. The pY-sCPBUE and pY-sCPBUEC plasmids were each introduced into the BY4741 strain, and in the case of pY-sCPBUEC, a second plasmid (pYH-sHB) containing a redox partner enzyme was co-transformed to create a total of three new strains.

[0408] 5-7. Preparation of solid media and seed culture media

[0409] The solid medium for obtaining yeast colonies transformed with one plasmid (pY-sCPBUE, pY-sCPBUEC) was SD-Ura agar medium lacking uracil, and the solid medium for obtaining yeast colonies co-transformed with two plasmids (pY-sCPBUEC / pY-sHB) was SD-His / -Ura agar medium lacking both uracil and histidine. To prepare the SD-Ura agar medium, 6.7 g of amino acid-free yeast nitrogen base and 0.77 g of -Ura DO (drop out) supplement were dissolved in 900 mL of distilled water, the pH was adjusted to approximately 5.6–6.0, and 20 g of agar was added to dissolve. To prepare SD-His / -Ura agar medium, 6.7 g of yeast nitrogen base without amino acids and 0.75 g of -His / -Ura DO (drop out) supplement were dissolved in 900 mL of distilled water, the pH was adjusted to approximately 5.6–6.0, and 20 g of agar was added and dissolved. The solid medium prepared as above was autoclaved at 121°C for 20 minutes, cooled, and 100 mL of 20% (w / v) glucose was added. A certain amount was poured into a petri dish, solidified at room temperature, and then stored in the refrigerator.

[0410] For the seed culture of strains transformed with one plasmid (pYES2, pY-sCPBU, pY-sCPBUD, pY-sCPBUDC, pY-sCPBUE, pY-sCPBUEC), SD-Ura medium was used, and for the seed culture of strains co-transformed with two plasmids (pY-sCPBUDC / pY-sHB, pY-sCPBUEC / pY-sHB), SD-His / -Ura medium was used. SD-Ura agar medium and SD-His / -Ura agar medium were prepared in the same manner as above, and the prepared seed culture medium was autoclaved at 121°C for 20 minutes, cooled, and then 100 mL of 20% (w / v) glucose was added. The medium was then stored at room temperature.

[0411] For all strains used in the test, expression was regulated by galactose using the Gal promoter, so galactose was added to the culture medium to induce expression. Therefore, for the main culture, a complex medium, YPDG medium, was used. To prepare the YPDG medium, 10 g of yeast extract and 20 g of peptone were dissolved in 900 mL of distilled water, autoclaved at 121℃ for 20 minutes, cooled, and then 50 mL of 20% (w / v) glucose and 50 mL of 20% (w / v) galactose were added. After preparation, it was stored at room temperature. Cholesterol was added to the culture medium so that the final concentration was 100 mg / L.

[0412] Culture was performed under the same conditions as in 2-3, with the main culture time being 72 hours. The main culture was performed more than three times to confirm reproducibility, and each culture was performed with two samples per condition (duplicate).

[0413] 5-8. Extraction

[0414] Step 2 and Step 3 material extraction was performed by centrifuging 50 mL of the culture medium at 3,000 g for 30 minutes after the completion of the cultivation, removing the supernatant, and using the yeast pellet obtained. To the recovered cell pellet, 20 mL of 50 mM Tris-HCl (pH 7.5) and approximately 1.0 to 1.5 g of glass beads were added, and the cells were disrupted by resuspension and vortexing. After cell disruption, extraction was performed once with 5 mL of ethyl acetate and twice with 3 mL. The recovered ethyl acetate supernatant was dried using a rotary vacuum concentrator to prepare an analysis sample.

[0415] 5-9. Analysis

[0416] The standard solution was prepared by taking 10 mg each of cholesterol, 7α-hydroxycholesterol, step 2 substances, and step 3 substances, diluting them in 10 mL of methanol, and then filtering them through a 0.22 μm filter. In this test, the test solution was prepared by diluting 50 mL of culture broth with 1 mL of methanol and then filtering them through a 0.22 μm filter (concentration ratio = 50).

[0417] The production of each substance was confirmed using the peak area of ​​the test solution obtained by testing the blank test solution (methanol), standard solution, and test solution under the operating conditions of Table 33.

[0418] LC System Column Waters symmetry C18 (4.6 x 75 mm, 3.5 μm) Column Temperature 25 °C Mobile Phase A 100% Methanol Mobile Phase B 100% Acetonitrile Injection Volume 10 μL Sample Temperature 4 °C Run Time 20 min Needle Wash 80% Methanol Seal Wash 10% Methanol Gradient Mode Time (min) Flow (mL / min) A (%) B (%) 0 0.6 2 0 8 0 8 0.6 2 0 8 0 8 5 1 0 8 0 2 0 1 6 5 1 0 8 0 2 0 1 7 1 0 8 0 2 0 1 7 1 0 8 0 2 0 1 0 8 0 MS System Ionization Source APCI Corona 4.0 μA Cone 25 V Capillary 4.0 kVA PCI Probe Temperature 350 °C Gas Desolvation Flow 300 L / hr RM Resolution 13.0HM Resolution 115.0Ion Energy 10.8RM Resolution 23.0HM Resolution 215.0Ion Energy 20.5Collision 2.0MRM Mode CompoundMother IonDaughter IonCholesterol396.3147.01 Step a Step 367.5241.02 b Step 401.3365.53 c 417.3269.0

[0419] a 7α-hydroxycholesterol; b 7α-hydroxy-4-cholesten-3-one; c 4-cholesten-7α,12α-diol-3-one

[0420] 5-10. Confirmation of two-stage material production and comparison of productivity using ERG26

[0421] The production and productivity of the second-stage material were confirmed using pY-sCPBUE, which has the ERG26 gene from Saccharomyces cerevisiae inserted. For productivity comparison, cultivation was performed using the empty vector pYES2, the first-stage material production plasmid pY-sCPBU, and the second-stage material production plasmid pY-sCPBUD containing the second-stage conversion gene HSD3B7 from H. sapiens.

[0422] As a result of confirming cell growth according to each plasmid, as shown in Figure 14a, pYES2 was the highest, and the cell growth amounts of the other three strains were confirmed to be at an equivalent level.

[0423] The productivity of the two-step material was confirmed through LC-MS / MS, and the average value and standard deviation were calculated using the results of four batches for each plasmid condition. The productivity according to each plasmid was confirmed to be 0.012 mg / L for pY-sCPBU, and 0.023 mg / L for pY-sCPBUD and pY-sCPBUE. It was confirmed that the productivity was improved by about two times under the condition using the synthetic ERG26 overexpression (pY-sCPBUE) compared to the condition using the existing ERG26 in yeast (pY-sCPBU). However, when the condition using the two-step conversion gene HSD3B7 derived from H. sapiens (pY-sCPBUD) and pY-sCPBUE were compared, they were confirmed to be at an equivalent level.

[0424] The extraction method used in this test was a 2-3 step target material extraction method, so the extraction efficiency of the 1st step material was not high. However, when comparing the production volumes under the assumption that the 1st step material extraction was performed under equivalent conditions, it was confirmed that the 1st step material productivity was the highest at 0.741 mg / L in pY-sCPBUE, as shown in Table 34 and Figure 14b.

[0425] No. Strain concentration (mg / L) Cholesterol Stage 1 a Step 2 b 1BY4741 (pYES2)0.694±0.268ND c ND2BY4741 (pY-sCPBU)4.133±0.8710.364±0.2270.011±0.0083BY4741 (pY-sCPBUD)5.681±1.4380.690±0.1520.023±0.0114BY4741 (pY-sCPBUE)5.997±1.9070.741±0.5500.023±0.005

[0426] a 7α-hydroxycholesterol; b 7α-hydroxy-4-cholesten-3-one; c 4-cholesten-7α,12α-diol-3-one

[0427] 5-11. Verification of three-stage material production and comparison of productivity using ERG26

[0428] Using two strains, pY-sCPBUEC and pY-sCPBUEC / pYH-sHB, into which the ERG26 gene from Saccharomyces cerevisiae was inserted, we aimed to further confirm the effect of the gene on the three-stage material productivity.

[0429] For productivity comparison, cultivation was performed using pYES2 and pY-sCPBU, and the three-stage material production plasmids pY-sCPBUDC and pY-sCPBUDC / pYH-sHB containing the two-stage conversion gene HSD3B7 derived from H. sapiens.

[0430] As a result of confirming cell growth according to each plasmid, as shown in Fig. 14c, pYES2 showed the highest cell growth, similar to the two-step material production confirmation test, and compared to pY-sCPBUDC and pY-sCPBUEC, it was confirmed that the cell growth of pY-sCPBUDC / pYH-sHB and pY-sCPBUEC / pYH-sHB, which contain one more plasmid, was slightly lower.

[0431] For the 3-step material, the productivity was also confirmed using LC-MS / MS, and the average value and standard deviation were calculated using the results of three batches for each plasmid condition. As a result, as shown in Table 35 and Fig. 14d, pY-sCPBUDC was confirmed to be 0.022 mg / L, pY-sCPBUDC / pYH-sHB was 0.030 mg / L, pY-sCPBUEC was 0.028 mg / L, and pY-sCPBUEC / pYH-sHB was 0.035 mg / L. Through these results, it was confirmed that the 3-step material production differed from a minimum of 0.022 mg / L to a maximum of 0.035 mg / L depending on the presence or absence of a coenzyme (redox partner) and the type of conversion gene used (HDS3B7, ERG26).

[0432] No. Strain concentration (mg / L) Cholesterol Stage 1 a Step 2 b Step 3 c 1BY4741 (pYES2)0.849±0.303ND dNDND2BY4741 (pY-sCPBU)4.875±0.4460.394±0.0270.005±0.008ND3BY4741 (pY-sCPBUDC)6.595±1.4330.465±0.0550.008±0.0040.022±0.0174BY4741 (pY-sCPBUDC / pYH-sHB)5.346±1.0850.555±0.0320.006±0.0030.030±0.0115BY4741 (pY-sCPBUEC)7.770±0.3790.657±0.0370.014±0.0050.028±0.0036BY4741 (pY-sCPBUEC / pYH-sHB)5.942±2.5910.604±0.2050.006±0.0060.035±0.009

[0433] a 7α-hydroxycholesterol; b 7α-hydroxy-4-cholesten-3-one; c 4-cholesten-7α,12α-diol-3-one; d Not detected

[0434] [Example 6]

[0435] Construction of a novel 7α-hydroxy-4-cholesten-3-one producing plasmid from mammalian genetic resources

[0436] To construct a CA biosynthetic pathway in yeast, the HSD3B7 gene, which is involved in the conversion of 7α-hydroxycholesterol to 7α-hydroxy-4-cholesten-3-one (a step 2 product), was obtained from the full form of H. sapiens (Transcript variant 1) and various mammalian (Rattus norvegicus, Oryctolagus cuniculus, Ursus maritimus, Bos Taurus, Gallus gallus) strains to construct a plasmid producing a step 2 product in the novel CA pathway.

[0437] 6-1. Template vector

[0438] pY-sCPBU, a plasmid for producing 7α-hydroxycholesterol from cholesterol, was used to insert a novel mammalian two-step substance production gene.

[0439] 6-2. Insert gene

[0440] The base sequences of the expression cassettes containing the newly synthesized HSD3B7 genes from H. sapiens, R. norvegicus, O. cuniculus, U. maritimus, B. Taurus, and G. gallus are shown in Figures 15a to 15f, respectively. The HSD3B7 gene from H. sapiens used in the existing two-step material production was transcript variant 2, which was a form in which some N-terminal sequences were deleted from the entire sequence of the HSD3B7 gene, and the newly introduced gene was transcript variant 1 in its entire form.

[0441] 6-3. Cloning primer

[0442] For plasmid construction, the expression cassette containing each gene was amplified using the primers in Table 36 and used to construct the target plasmid.

[0443] Cloning primer direction sequence size (bp) sequence number CAUP_SalI_F forward 5'- gaaagccggcgtcgacacggattagaagccgccgag -3'36 bp 105 CAUP_SacII_r reverse 5'- gcttctaatccgtccgcggaaagccttcgagcgtccc -3'37 bp 106

[0444] 6-4. Construction of a novel 7α-hydroxy-4-cholesten-3-one production plasmid

[0445] An expression cassette containing six novel mammalian HSD3B7 genes was cleaved at the SalI and SacII restriction enzyme sites and inserted into the pY-sCPBU plasmid treated with the same restriction enzymes. The six constructed plasmids were named pY-sCPBUD(Hv), pY-sCPBUD(R), pY-sCPBUD(O), pY-sCPBUD(U), pY-sCPBUD(B), and pY-sCPBUD(G), respectively, and the construction schematic is as shown in Figure 16.

[0446] To determine whether the plasmids were properly constructed, plasmid DNA fragmentation was performed. Six newly constructed plasmids were subjected to DNA fragmentation using the restriction enzyme ClaI. The expected fragment sizes upon digestion are shown in Table 37. As shown in Figure 17, the experimental results confirmed that the plasmids were of the appropriate size.

[0447] Plasmid NameClone SizeExpected SizepY-sCPBUD(Hv)16,603 bp8,484 bp / 4,900 bp / 3,219 bppY-sCPBUD(R)16,510 bp8,484 bp / 3,219 bp / 2,731 bp / 2,076 bppY-sCPBUD(O)16,606 bp8,484 bp / 4,903 bp / 3,219 bppY-sCPBUD(U)16,423 bp8,484 bp / 4,720 bp / 3,219 bppY-sCPBUD(B)16,600 bp8,484 bp / 4,897 bp / 3,219 bppY-sCPBUD(G)16,408 bp8,484 bp / 4,705 bp / 3,219 bp

[0448] [Example 7]

[0449] Production of 7α-hydroxy-4-cholesten-3-one from various mammalian genetic sources using yeast.

[0450] During the construction of the CA biosynthetic pathway, the second step material conversion gene was HSD3B7, and its production was confirmed using the transcript variant 2 of H. sapiens. By replacing it with the full form of H. sapiens (transcript variant 1) and the HSD3B7 gene derived from various mammals (Rattus norvegicus, Oryctolagus cuniculus, Ursus maritimus, Bos taurus, Gallus gallus), we attempted to confirm the production of 7-hydroxy-4-cholesten-3-one.

[0451] The production strain is Saccharomyces cerevisiae BY4741, and the plasmid information is as shown in Table 38.

[0452] Plasmid details pYES2P gal1 E. coliand yeast shuttle vector, 2μ origin,URA3gene, Amp rpY-sCPBUpYES2 containing the codon-optimizedCYP7A1, POR(Cytochrome P450 oxidoreductase) andCYB5(Cytochrome b5) gene fromHomo sapiens,andUPC2-1gene fromSaccharomyces cerevisiaepY-sCPBUDpY-sCPBU containingHSD3B7gene(transcript variant 2) fromH. sapienspY-sCPBUD(Hv)pY-sCPBU containing the codon-optimizedHSD3B7gene(Full form, transcript variant 1) fromH. sapienspY-sCPBUD(R)pY-sCPBU containing the codon-optimizedHSD3B7gene fromRattus norvegicuspY-sCPBUD(O)pY-sCPBU containing the codon-optimizedHSD3B7gene fromOryctolagus cuniculuspY-sCPBUD(U)pY-sCPBU containing the codon-optimizedHSD3B7gene fromUrsus maritimuspY-sCPBUD(B)pY-sCPBU containing the codon-optimizedHSD3B7gene fromBos tauruspY-sCPBUD(G)pY-sCPBU containing the codon-optimizedHSD3B7gene fromGallus gallus

[0453] 7-1. Introduction of expression plasmid into yeast

[0454] In order to introduce the six new plasmids constructed in Example 5 into yeast, Sc EasyComp from Invitrogen was used. TM A transformation kit was used. The transformation method was carried out according to the protocol in the kit, and transformation was performed in the same manner as in Example 3-1.

[0455] The medium and reagents were prepared and used in the same manner as in Example 1-2, and the culture was conducted in the same manner as in 1-3. At this time, cholesterol was added to the culture medium so that the final concentration was 100 mg / L.

[0456] 7-2. Extraction

[0457] 50 mL of the main culture was centrifuged at 3,000 g for 30 minutes, the supernatant was removed, and 20 mL of 50 mM Tris-HCl (pH 7.5) and 1 to 1.5 g of glass beads were added to the obtained yeast pellet, and the mixture was vortexed for 5 minutes to disrupt the cells. 5 mL of ethyl acetate was added to separate the layers. To ensure good layer separation, centrifugation was performed at 3,000 g for 10 minutes, and the upper ethyl acetate layer was recovered. This same recovery process was repeated twice using 3 mL of ethyl acetate, and the recovered ethyl acetate supernatant was dried using a rotary vacuum concentrator.

[0458] 7-3. Analysis

[0459] The standard solution was prepared by taking 10 mg each of cholesterol, 7α-hydroxycholesterol, and the second-phase material, diluting them in 10 mL of methanol, and filtering them through a 0.22 μm filter. In this test, the test solution was prepared by diluting 50 mL of culture broth with 1 mL of methanol, concentrating and drying the sample, and centrifuging (12,000 rpm, RT for 10 minutes). The supernatant was then collected and used for evaluation.

[0460] The production of each substance was confirmed using the peak area of ​​the test solution obtained by testing the blank test solution (methanol), standard solution, and test solution under the operating conditions of Table 16. At this time, under the conditions of Table 16, the execution time was changed to 20 minutes, and RM resolution 1 and RM resolution 2 were each changed to 15.0.

[0461] 7-4. Productivity verification of strains containing the HSD3B7 gene from various mammals.

[0462] After transformation of yeast with the empty vector pYES2, the existing 2-step target substance production plasmid pY-sCPBUD (transcript variant 2 of H. sapiens), and 2-step target substance production plasmids constructed from the origin of 6 new species (pY-sCPBUD(Hv), pY-sCPBUD(R), pY-sCPBUD(O), pY-sCPBUD(U), pY-sCPBUD(B), pY-sCPBUD(G)), the production of the 2-step target substance was confirmed by culturing.

[0463] The culture was performed twice to identify trends and ensure reproducibility, and the average value of the two culture results was calculated. As shown in Figure 18a, in the case of the main culture cell growth, the OD of the empty vector pYES2 after the final culture 600nm While the OD of the 6 plasmids producing the second stage target substance is approximately 43 600nm It was confirmed that cell growth was generally lower than that of the empty vector, at approximately 36 to 39.

[0464] As a result of confirming the productivity of the second-stage target substance, as shown in Fig. 18b and Table 39, in the case of the existing second-stage target substance production plasmid, pY-sCPBUD, it was confirmed that the second-stage target substance was produced at approximately 0.018 mg / L. In the case of the new plasmids, pY-sCPBUD(R) derived from R. norvegicus, pY-sCPBUD(U) derived from U. maritimus, and pY-sCPBUD(G) derived from G. gallus were confirmed to be at an equivalent level of 0.017 to 0.020 mg / L. The conditions for increased productivity compared to the existing strains were the conditions containing a total of three new genes; pY-sCPBUD(Hv) using the full form of H. sapiens produced 1.184 mg / L, pY-sCPBUD(O) derived from O. cuniculus produced 1.166 mg / L, and pY-sCPBUD(Hv) derived from B. pY-sCPBUD(B) derived from Taurus was confirmed to be 1.494 mg / L, confirming an approximately 80-fold increase in productivity compared to the existing strain. In addition, in the case of strains with improved productivity of the second-stage target substance, it was confirmed that the production of the precursor 7α-hydroxycholesterol was reduced.

[0465] No. Strain concentration (mg / L) Cholesterol 7α-hydroxycholesterol 7α-hydroxy-4-cholesten-3-one 1BY4741 (pYES2) 1.016±0.118ND aND2BY4741 (pY-sCPBUD)7.074±0.5960.832±0.0720.018±0.0033BY4741 (pY-sCPBUD(Hv))7.468±2.9360.050±0.0211.184±0.6564BY4741 (pY-sCPBUD(R))7.159±0.5820.975±0.0570.017±0.0015BY4741 (pY-sCPBUD(O))8.408±0.4900.094±0.0831.166±0.1536BY4741 (pY-sCPBUD(U))6.712±0.5180.995±0.0700.020±0.0037BY4741 (pY-sCPBUD(B))9.399±1.3770.063±0.0031.494±0.2268BY4741 (pY-sCPBUD(G))7.495±0.8971.099±0.0480.022±0.004

[0466] a Not detected

[0467] 7-5. Final reproducibility confirmation and candidate strain selection for 7α-hydroxy-4-cholesten-3-one acid

[0468] Through primary and secondary cultures, three candidate strains with improved productivity compared to the conditions using the existing plasmid (pY-sCPBUD) were secured. The tertiary culture was conducted on pY-sCPBUD(Hv), pY-sCPBUD(O), and pY-sCPBUD(B), which were confirmed to have improved productivity compared to the existing strains, to ensure reproducibility of the primary and secondary culture results and to select the final candidate strains.

[0469] As shown in Fig. 18c, similar to the results of the preceding total secondary culture, it was confirmed that the cell growth rate was generally lower for the strain containing the plasmid for producing the second-stage target substance compared to the empty vector pYES2.

[0470] As a result of the analysis, as shown in Table 40, the existing pY-sCPBUD was approximately 0.038 mg / L, pY-sCPBUD(Hv) was 1.780 mg / L, pY-sCPBUD(O) was 1.365 mg / L, and pY-sCPBUD(B) was 1.640 mg / L, showing the same pattern as the previous results.

[0471] No. Strain concentration (mg / L) Cholesterol 7α-hydroxycholesterol 7α-hydroxy-4-cholesten-3-one 1BY4741 (pYES2) 1.009±0.045ND a ND2BY4741 (pY-sCPBUD)6.390±0.0880.666±0.0080.038±0.0003BY4741 (pY-sCPBUD(Hv))10.785±1.0010.076±0.0001.780±0.1944BY4741 (pY-sCPBUD(O))9.286±0.5040.052±0.0021.365±0.0135BY4741 (pY-sCPBUD(B))11.072±0.4480.058±0.0021.640±0.040

[0472] a Not detected

[0473] Accordingly, the average value was calculated and confirmed using the overall culture results. As a result, as shown in Table 41 and Fig. 18d, the existing pY-sCPBUD was approximately 0.024 mg / L, pY-sCPBUD(Hv) was 1.383 mg / L, pY-sCPBUD(O) was 1.232 mg / L, and pY-sCPBUD(B) was 1.552 mg / L, confirming that the productivity was improved by approximately 60 times compared to the productivity of the existing strain.

[0474] No. Strain concentration (mg / L) Cholesterol 7α-hydroxycholesterol 7α-hydroxy-4-cholesten-3-one 1BY4741 (pYES2) 1.013±0.100ND aND2BY4741 (pY-sCPBUD)6.846±0.5860.777±0.0980.024±0.0103BY4741 (pY-sCPBUD(Hv))8.574±2.9200.059±0.0211.383±0.6154BY4741 (pY-sCPBUD(R))7.159±0.5820.975±0.0570.017±0.0015BY4741 (pY-sCPBUD(O))8.701±0.6450.080±0.0701.232±0.1566BY4741 (pY-sCPBUD(U))6.712±0.5180.995±0.0700.020±0.0037BY4741 (pY-sCPBUD(B))10.068±1.3750.061±0.0041.552±0.1918BY4741 (pY-sCPBUD(G))7.495±0.8971.099±0.0480.022±0.004

[0475] a Not detected

[0476] [Example 8]

[0477] Construction of a novel 7α-hydroxy-4-cholesten-3-one production plasmid from microbial genetic resources

[0478] The second step product produced from cholesterol in the CA biosynthetic pathway in yeast is 7α-hydroxy-4-cholesten-3-one. The enzyme is 3β-hydroxysteroid dehydrogenase. A gene with the same function was selected and synthesized from a microbial source, and the selected gene was inserted into a plasmid producing 7α-hydroxycholesterol (CAS: 566-26-7, the first step product) to construct a plasmid producing the second step product in the novel CA pathway.

[0479] 8-1. Template vector

[0480] pY-sCPBU, a plasmid for producing 7α-hydroxycholesterol from cholesterol, was used to insert a novel mammalian two-step substance production gene.

[0481] 8-2. Insert gene

[0482] 3β-hydroxy-Δ, a two-step transition gene 5 -C 27 Steroid oxidoreductase (3β-hydroxy-Δ 5 -C 27 Steroid oxidoreductase) catalyzes the 7α-hydroxycholesterol, a step 1 product of the CA pathway, as shown in Figure 19. 5 -3 hydroxy steroids Δ 4 -3 An enzyme that catalyzes the oxidative conversion to the keto form, producing 7α-hydroxy-4-cholesten-3-one. We sought to identify genes from microorganisms that have the same enzymatic reaction as this function, and these were mainly genes that react similarly to substances with the same substrate backbone (e.g., cholesterol) in the cholesterol degradation or oxidation pathway.

[0483] The corresponding pathway and related genes were selected from six genes in total from Mycobacterium tuberculosis H37Rv, Brevibacterium sterolicum, Saccharomyces cerevisiae, and Lactobacillus brevis, and the selected genes were synthesized by codon optimization in yeast. The synthesized genes were delivered in the form of insertion into the pYES2 vector so that they are expressed by the Gal promoter, and the corresponding base sequences are as shown in Figures 20a to 20f.

[0484] 8-3. Cloning primer

[0485] To construct six types of plasmids, the expression cassettes containing each gene were amplified using the primers in Table 40 and used to construct the target plasmids.

[0486] Cloning primer direction sequence size (bp) sequence number CAUP_SalI_F forward 5'- gaaagccggcgtcgacacggattagaagccgccgag -3'36 bp 107 CAUP_SacII_r reverse 5'- gcttctaatccgtccgcggaaagccttcgagcgtccc-3'37 bp 108

[0487] 8-4. Construction of a novel 7α-hydroxy-4-cholesten-3-one production plasmid

[0488] An expression cassette containing six novel microbial-derived 3β-hydroxysteroid dehydrogenase genes was cleaved at the SalI and SacII restriction enzyme sites and inserted into the pY-sCPBU plasmid treated with the same restriction enzymes. The six constructed plasmids were named pY-sCPBUMhD, pY-sCPBUMcD, pY-sCPBUBcB, pY-sCPBUBcB(woSS), pY-sCPBUSsR, and pY-sCPBULhD, respectively, and the construction schematic is as shown in Figure 21.

[0489] To determine whether the plasmids were properly constructed, plasmid DNA fragmentation was performed. The results of plasmid DNA fragmentation are as follows. Six newly constructed plasmids were subjected to DNA fragmentation using restriction enzymes NcoI, NdeI, or ClaI. The expected fragment sizes upon digestion are shown in Table 43. As shown in Figure 22, the experimental results confirmed that all of them were suitable for the corresponding sizes.

[0490] Plasmid NameClone SizeRestriction EnzymeExpected SizepY-sCPBUMhD16,606 bpNcoI6,604 bp / 5,241 bp / 4,761 bppY-sCPBUMcD17,230 bpNcoI6,604 bp / 5,865 bp / 4,761 bppY-sCPBUBcB17,152 bpNcoI6,604 bp / 4,761 bp / 4,005 bp / 1,782 bppY-sCPBUBcB(woSS)17,020 bpNcoI6,604 bp / 4,761 bp / 3,873 bp / 1,782 bppY-sCPBUSsR16,423 bpNdeI9,392 bp / 3,950 bp / 3,081 bppY-sCPBULhD16,267 bpClaI8,484 bp / 4,564 bp / 3,219 bp

[0491] [Example 9]

[0492] Production of 7α-hydroxy-4-cholesten-3-one from microbial genetic resources using yeast

[0493] In the process of constructing the CA biosynthetic pathway, the conversion gene for 7α-hydroxy-4-cholesten-3-one, a second-step substance, was replaced with genes from various microorganisms to confirm the production of 7α-hydroxy-4-cholesten-3-one.

[0494] 9-1. Production strains and plasmids

[0495] The production strain is Saccharomyces cerevisiae BY4741, and the plasmid information is as shown in Table 44.

[0496] Plasmid details pYES2P gal1 E. coliand yeast shuttle vector, 2μ origin,URA3gene, Amp r pY-sCPBUDpY-sCPBU 1containing the codon-optimizedHSD3B7gene fromHomo sapienspY-sCPBUMhDpY-sCPBU containing the codon-optimizedhsdDgene fromMycobacterium tuberculosisH37RvpY-sCPBUMcDpY-sCPBU containing the codon-optimizedchoDgene fromM. tuberculosisH37RvpY-sCPBUBcBpY-sCPBU containing the codon-optimizedchoBgene fromBrevibacterium sterolicumpY-sCPBUBcB(woSS)pY-sCPBU containing the codon-optimizedchoB(w / o signal peptide) gene fromB. sterolicumpY-sCPBUSsRpY-sCPBU containing the codon-optimizedYKL107Wgene fromS. cerevisiaepY-sCPBULhDpY-sCPBU containing the codon-optimized3BHSDgene fromLactobacillus brevis

[0497] 1 pY-sCPBU: pYES2 containing codon-optimized CYP7A1, POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5) genes derived from Homo sapiens and UPC2-1 gene derived from Saccharomyces cerevisiae

[0498] 9-2. Introduction of expression plasmid into yeast

[0499] To introduce each plasmid in Table 43 into yeast, Sc EasyComp from Invitrogen was used. TM A transformation kit was used. The transformation method was carried out according to the protocol in the kit, and transformation was performed in the same manner as in Example 4-1.

[0500] The medium and reagents were prepared and used in the same manner as in Example 2-2, and the culture was conducted in the same manner as in 2-3. At this time, cholesterol was added to the culture medium so that the final concentration was 100 mg / L, and the main culture was conducted for 72 hours at 30°C and 180 rpm after inoculation.

[0501] 9-3. Extraction

[0502] 50 mL of the main culture was centrifuged at 3,000 g for 30 minutes, the supernatant was removed, and 20 mL of 50 mM Tris-HCl (pH 7.5) and 1 to 1.5 g of glass beads were added to the obtained yeast pellet, vortexed for 5 minutes to disrupt the cells, and 5 mL of ethyl acetate was added to separate the layers. To ensure good layer separation, centrifugation was performed at 3,000 g for 10 minutes, and the upper ethyl acetate layer was recovered. This same recovery process was repeated twice using 3 mL of ethyl acetate, and the recovered ethyl acetate supernatant was dried using a rotary vacuum concentrator.

[0503] 9-4. Analysis

[0504] The standard solution was prepared by taking 10 mg each of cholesterol, 7α-hydroxycholesterol, 5-cholesten-3-one and 4-cholesten-3-one, which have the possibility of conversion due to the 3β-hydroxysteroid dehydrogenase gene of phase 2 substances and microorganisms, diluting them in 10 mL of methanol, and filtering them through a 0.22 μm filter. In this test, the test solution was prepared by diluting 50 mL of culture broth with 1 mL of methanol and then centrifuging (12,000 rpm, RT condition for 10 minutes). The supernatant was then collected and used for evaluation.

[0505] The production of each substance was confirmed using the peak area of ​​the test solution obtained by testing the blank test solution (methanol), standard solution, and test solution under the operating conditions of Table 34. At this time, the MRM mode is as shown in Table 45.

[0506] MRM Mode Compound Mother Ion Daughter Ion Cholesterol 396.4147.07α-Hydroxycholesterol 367.5241.07α-Hydroxy-4-cholesten-3-one 401.3383.55-cholesten-3-one 385.3109.04-cholesten-3-one 385.4123.1

[0507] 9-5. Productivity of strains expressing the 3β-hydroxysteroid dehydrogenase gene derived from various microorganisms

[0508] The 2-step target substance production plasmids (pY-sCPBUMhD, pY-sCPBUMcD, pY-sCPBUBcB, pY-sCPBUBcB(woSS), pY-sCPBUSsR, pY-sCPBULhD) constructed by applying the empty vector pYES2, the existing 2-step target substance production plasmid pY-sCPBUD (transcript variant 2 of H. sapiens), and the 3β-hydroxysteroid dehydrogenase gene from microorganisms were transformed into yeast and cultured to confirm the production of the 2-step target substance.

[0509] Cultivation was performed in a 60 mL scale in a 250 mL baffled flask, and each condition was performed twice. A total of two cultivations were performed to confirm productivity and ensure reproducibility of the test. The average value for the two batches of each strain was calculated, and as shown in Figure 23a, the OD of the empty vector pYES2 600nm While the OD of the six new 2-step target substance production plasmids is approximately 43 600nm It was confirmed that cell growth was generally lower than that of the empty vector, at approximately 37 to 41.

[0510] As a result of confirming the productivity of the second-stage target substance, as shown in Table 46 and Figures 23b and 23c, in the case of the existing second-stage target substance production plasmid, pY-sCPBUD, it was confirmed that the second-stage target substance was produced at approximately 0.015 mg / L. The second-stage target substance production of the new plasmid was confirmed as 0.020 mg / L for pY-sCPBUMhD, 0.018 mg / L for pY-sCPBUMcD, 0.021 mg / L for pY-sCPBUBcB, 0.026 mg / L for pY-sCPBUSsR, and 0.031 mg / L for pY-sCPBULhD, and no production was confirmed for pY-sCPBUBcB(woSS) without a signal peptide. The productivity of 5-cholesten-3-one, which can be produced by the 3β-hydroxysteroid dehydrogenase gene from microorganisms, was confirmed to be almost the same at 0.008~0.010 mg / L under all conditions except the pY-sCPBUMhD condition. In the case of pY-sCPBUMhD, the productivity of 5-cholesten-3-one was confirmed to be 0.026 mg / L, which was about 3 times higher than that of the other conditions, and the productivity of 7α-hydroxycholesterol was confirmed to be low at 0.024 mg / L, compared to 0.471 mg / L of the existing strain. In the condition using the pY-sCPBUBcB(woSS) plasmid, the productivity of all target substances was confirmed to be generally low. 4-cholesten-3-one, another substance that can be produced by microbial genes, was not confirmed under any conditions.

[0511] No. Strain concentration (mg / L) Cholesterol 7α-hydroxycholesterol 7α-hydroxy-4-cholesten-3-one 5-cholesten-3-one 4-cholesten-3-one 1BY4741 (pYES2) 0.423±0.064ND aND0.008±0.000ND2BY4741 (pY-sCPBUD)3.075±0.7020.471±0.1030.015±0.0030.009±0.001ND3BY4741 (pY-sCPBUMhD)2.137±0.6170.024±0.0070.020±0.0050.026±0.004ND4BY4741 (pY-sCPBUMcD)3.299±1.1150.684±0.2200.018±0.0040.009±0.001ND5BY4741 (pY-sCPBUBcB)4.512±0.6400.921±0.0810.021±0.0010.009±0.001ND6BY4741 (pY-sCPBUBcB(woSS)0.572±0.0490.036±0.004ND0.008±0.000ND7BY4741 (pY-sCPBUSsR)4.826±0.4550.998±0.0650.026±0.0010.010±0.001ND8BY4741 (pY-sCPBULhD)5.202±0.2551.128±0.0510.031±0.0010.010±0.000ND

[0512] a Not detected

[0513] The results of two cultures were combined and averaged to determine the cell growth rate, which was lower when the plasmid containing the two-step target substance production was included compared to the empty vector pYES2.

[0514] The productivity of the second-stage target substance was very low at 0.015 mg / L in the case of pY-sCPBUD using the existing HSD3B7 gene (transcript variant 2) derived from H. sapiens. Through this test, it was confirmed that the productivity of the second-stage target substance was improved by approximately 2-3 times under all conditions except the pY-sCPBUBcB(woSS) condition by applying alternative genetic resources derived from various microorganisms.

[0515] The results of this test confirmed an alternative route for producing step 2 materials of the CA pathway using microbial genes other than the existing mammalian-derived route.

[0516] [Example 10]

[0517] Confirmation of production of 7α,12α-dihydroxycholest-4-en-3-one from cholesterol using yeast.

[0518] We aimed to confirm the production of 7α,12α-dihydroxycholest-4-en-3-one (CAS No. 1254-03-1), a step 3 product of the 15-step biosynthetic pathway for producing CA using cholesterol as a substrate in yeast.

[0519] 10-1. Introduction of expression plasmid into yeast

[0520] The production strain used was Saccharomyces cerevisiae BY4741 (genotype; MATa his3Δ1 leu2Δ met15Δ ura3Δ), and the plasmid information is as shown in Table 47.

[0521] Plasmid details (see pYES2P) gal1 E. coli and yeast shuttle vector, 2μ origin, URA3 gene, Amp r Negative control pYES_HISP gal1 E. coli and yeast shuttle vector, 2μ origin, HIS3 gene, Amp rpY-sCPBUpYES2 containing the codon-optimized CYP7A1, POR(Cytochrome P450 oxidoreductase), and CYB5(Cytochrome b5) from H. sapiens and UPC2-1 gene from S. cerevisiae1 단계 생산플라스미드pY-sCPBUDpY-sCPBU containing the codon-optimized HSD3B7 gene from Homo sapiens (이하 H. sapiens)본 실시예pY-sCPBUDCpY-sCPBUD containing the codon-optimized CYP8B1 gene from H. sapienspYH-sHsPORpYES2_HIS containing the codon-optimized POR(Cytochrome P450 oxidoreductase) gene from H. sapienspYH-sOcPORpYES2_HIS containing the codon-optimized POR(Cytochrome P450 oxidoreductase) gene from Oryctolagus cuniculus (이하 O. cuniculus)pYH-sScNcp1pYES2_HIS containing the codon-optimized Ncp1 gene from S. cerevisiaepYH-sHBpYH-sHsPOR containing the codon-optimized CYB5(Cytochrome b5) gene from H. sapienspYH-sOBpYH-sOcPOR containing the codon-optimized CYB5(Cytochrome b5) gene from H. sapienspYH-sSBpYH-sScNcp1 containing the codon-optimized CYB5(Cytochrome b5) gene from H. sapiens

[0522] The BY4741 yeast strain was transformed with an empty vector, a 7α-hydroxycholesterol production plasmid, and 11 candidate plasmids for producing step 2 and 3 products, pYES2, pYES_HIS, pY-sCPBU, pY-sCPBUD, pY-sCPBUDC, pYH-sHsPOR, pYH-sOcPOR, pYH-sScNcp1, pYH-sHB, pYH-sOB, and pYH-sSB, to suitably produce the target substances. The transformation method was performed in the same manner as in Example 3-1. The yeast strains constructed to confirm step 2 and 3 products by combining the 11 plasmids above with the yeast strain BY4741 are as shown in Table 48.

[0523] No. Strain Plasmid I Plasmid II Reference 1 S. cerevisiae BY4741pYES2 - Negative control 2 pYES_HIS3pY-sCPBU-7α-hydroxycholesterol producing strain 4 pYES_HIS5pY-sCPBUD - Candidate strain for producing step 2 convertants, this example 6 pYES_HIS7pY-sCPBUDC - Candidate strain for producing step 3 convertants, this example 8 pYES_HIS9pYH-sHsPOR10pYH-sOcPOR11pYH-sScNcp112pYH-sHB13pYH-sOB14pYH-sSB

[0524] 10-2. Preparation of media and reagents

[0525] The solid medium for colony selection after transforming the yeast with the plasmid was SD-Ura agar or SD-Ura, His agar medium lacking uracil or uracil and histidine. To prepare it, dissolve 6.7 g of yeast nitrogen base without amino acids and 0.77 g of -Ura DO (drop out) supplement or 0.75 g of -His / -Ura DO (drop out) supplement in 900 mL of distilled water, adjust the pH to approximately 5.6-6.0, then add 20 g of agar and dissolve. Sterilize by autoclaving at 121℃ for 20 minutes, cool, add 100 mL of 20% glucose, and pour 20 mL each into petri dishes, solidify at room temperature, and then store in the refrigerator.

[0526] The seed culture medium was prepared by dissolving 6.7 g of yeast nitrogen base without amino acids and 0.77 g of -Ura DO(drop out) supplement or 0.75 g of -His / -Ura DO(drop out) supplement in 900 mL of distilled water, which is the same as the solid medium. The pH was adjusted to 5.6-6.0, autoclaved at 121℃ for 20 minutes, cooled, and then adding 100 mL of 20% glucose. After preparation, it was stored at room temperature. For the main culture, YPDG complex medium was used. Since the test condition was to add cholesterol as a substrate during the main culture, a 5% cholesterol stock was used and added to the culture medium at a final concentration of 100 mg / L for culture. The culture was performed under the same conditions as 2-3, but the main culture time was different, 72 hours.

[0527] 10-3. Extraction

[0528] To confirm the productivity of the CA pathway 2 and 3 step products, the same extraction method as in Example 4-2 was performed.

[0529] 10-4. Analysis

[0530] Standard and test solutions were prepared in the same manner as in Example 2-5, except that a 0.22 μm filter was used. The blank test solution (methanol), standard solution, and test solution were tested under the two operating conditions in Tables 49 and 50 below, and the production of each substance was confirmed using the peak areas of the test solutions obtained.

[0531] LC System Column Waters symmetry C18 (4.6 x 75 mm, 3.5 mm) Column temperature 25°C Mobile phase A 100% MeOH Mobile phase B 100% ACN Injection volume 2 mL Sample temperature 4°C Autosampler Extraction rate 5.0 mL / s Run time 45 min Needle wash 80% methanol Seal wash 10% methanol Gradient mode Time (min) Flow (mL / min) A (%) B (%) 0 0.32 0 8 0 1 8 0.32 0 8 0 18 11 2 8 0 20 4 0 0.32 0 8 0 45 0.32 0 8 0 MS System Ionization source APCI Polarity Positive Corona 4.0 mA Evaporator temperature 350°C Gas temperature 270°C Gas flow 12 L / min Nebulizer 60 psig Capillary 4000 V Fragment voltage 150 VMS scan range 200 to 600 m / z

[0532] LC System Column Waters symmetry C18 (4.6 x 75 mm, 3.5 mm) Column Temperature 25°C Mobile Phase A 100% MeOH Mobile Phase B 100% ACN Injection Volume 10 mL Sample Temperature 4°C Autosampler Extraction Rate 5.0 mL / s Run Time 45 min Needle Wash 80% Methanol Sealing Wash 10% Methanol Gradient Mode Time (min) Flow (mL / min) A (%) B (%) 0 0.6 2 0 8 0 8 0.6 2 0 8 0 8 5 1 0 8 0 2 0 1 6 5 1 0 8 0 2 0 1 7 1 0 8 0 2 0 1 0 8 0 2 0 1 0 8 0 MS System Ionization Source APCI Corona 4.0 mACone 25 V APCI Probe Temperature 350°C Gas Desolvation Flow 400 L / hr RM Resolution 12.9 HM Resolution 115 Ion Energy 10.8RM Resolution 22.8HM Resolution 215Ion Energy 20.5MRM Mode CompoundParent ionDaughter ionCholesterol396.3147.07α-Hydroxycholesterol383.5247.07α-Hydroxy-4-cholesten-3-one410.3383.57α,12α-Hydroxycholest-4-en-3-one417.3269.0

[0533] 10-5. Quantitative analysis of the target substance in Step 3

[0534] We aimed to compare the productivity of target substances in stages 2 and 3 of 14 candidate strains. Information on the cultured strains is provided in Table 48. To compare productivity, two duplicates were constructed for each sample, and two batches of cultures were performed to calculate the average value. Cell growth during culture is shown in Figure 24. Cell growth was confirmed to be relatively equivalent, with no differences in the presence or absence of gene introduction within the plasmid or in the gene combination.

[0535] The results of the productivity comparison for the target substances at each stage are shown in Table 48. The analysis results confirmed that both the 2nd and 3rd stage substances were produced in the newly constructed candidate strain. Since two plasmids with different auxotrophic markers were used for the production of the 3rd stage substance, pYES_HIS was introduced into yeast along with the production plasmid to determine the effect on productivity. As a result, it was confirmed that the productivity of the target substances at each stage tended to decrease slightly when the pYES_HIS empty vector was co-expressed, but it was confirmed that the difference in values ​​did not significantly affect the final result. The production of the 2nd stage substance was confirmed to be approximately 14 ㎍ / L even in the strain containing the plasmid for the 1st stage substance production (pY-sCPBU), and approximately 16 ㎍ / L was produced in the strain containing the candidate plasmid for the 2nd stage substance production (pY-sCPBUD). Although there was a small increase upon the amplification of the 2nd stage transgene, the increase was not considered to be significant. In the strain containing the plasmid for production of the 3-step material (pY-sCPBUDC), the productivity of the 3-step material was confirmed to be approximately 29.5 ㎍ / L, and in the case of the 2-step material, it was confirmed that it was converted to the 3-step material and the productivity was slightly lower compared to the strain containing the plasmid for production of the 2-step material (pY-sCPBUD).

[0536] And as a result of introducing redox partners from various origins for functional expression of the CYP8B1 gene, a 3-step material conversion gene, it was confirmed that the 3-step material productivity was improved more when the CYB5 (Cytochrome b5) gene, a cytochrome b5 gene, was simultaneously introduced than when only the NADPH-cytochrome P450 oxidoreductase was introduced (pYH-sHsPOR, pYH-sOcPOR, pYH-sScNcp1). Among them, the strain that simultaneously introduced the 3-step material conversion plasmids pY-sCPBUD and pYH-sHB had the highest 3-step material productivity of approximately 55.1 ㎍ / L, and this was finally selected as a 3-step material producing strain (Table 51).

[0537] No. Strain Plasmid I Plasmid II Concentration ppm (mg / L) ppb (μg / L) Cholesterol Step 1 Step 2 Step 3 Step 1 BY4741pYES2-1.0NDNDND2pYES_HIS1.1N.DNDND3pY-sCPBU-6.00.414.1ND4pYES_HIS5.50.410.3N.D.5pY-sCPBUD-7.80.616.0ND6pYES_HIS5.60.34.5N.D.7pY-sCPBUDC-7.60.47.229.58pYE S_HIS6.10.33.520.79pYH-sHsPOR9.40.56.049.910pYH-sOcPOR8.40.310.037.711pYH-sScNcp1 8.60.55.539.512pYH-sHB10.20.68.855.113pYH-sOB9.60.47.941.414pYH-sSB10.10.59.444.0

[0538] As a result of confirming the productivity of each step substance using a total of 10 candidate strains for producing 2nd and 3rd steps substances, it was confirmed that the strain composed of the 2nd step conversion gene HSD3B7 derived from H. sapiens, the 3rd step conversion gene CYP8B1, and the redox partner enzymes POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5) genes was the optimal combination for conversion to 3rd step substances. In the case of the 3rd step conversion gene CYP8B1, it was confirmed that the target substance was produced more efficiently when NADPH-cytochrome P450 oxidoreductase and cytochrome b5 were expressed simultaneously. This result is the same as the production of 7α-hydroxycholesterol, which is the 1st step substance. It was confirmed once again that the redox partner enzyme that transfers the electrons required for the enzymatic reaction is an essential element when expressing cytochrome P450 family enzymes known to be difficult to express in microorganisms. Additionally, the test results confirmed that the expression of the redox partner derived from H. sapiens, which is identical to the cytochrome P450 enzyme, was the most efficient, which is also the same as the results of the first-stage material production.

[0539] [Example 11]

[0540] Construction of a plasmid for producing 7α,12α-dihydroxycholest-4-en-3-one within a novel CA pathway from mammalian genetic resources.

[0541] To construct a CA biosynthetic pathway in yeast, CYP8B1 and its redox partner enzymes involved in the production of 7α,12α-dihydroxycholest-4-en-3-one (CAS No. 1254-03-1), a three-step product, were obtained from various mammalian sources to construct a three-step product production plasmid.

[0542] 11-1. Template vector

[0543] To insert CYP8B1, a novel mammalian 3-step substance-producing gene, pY-sCPBUD(B) and pY-sCPBUD(Hv), plasmids for 2-step substance production from cholesterol, were used. In addition, the genes for POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5), redox partner enzymes of the same origin as the 3-step conversion gene, were inserted into the pYES2_HIS vector.

[0544] 11-2. Insert gene

[0545] A.CYP8B1 gene

[0546] The base sequences of expression cassettes containing the CYP8B1 genes derived from R. norvegicus, O. cuniculus, B. Taurus, and G. gallus, which were newly synthesized by codon optimization in yeast, are shown in Figures 25a to 25d, respectively. The base sequence of the CYP8B1 gene derived from H. sapiens is shown in Figure 25e.

[0547] B.POR (Cytochrome P450 oxioreductase) gene

[0548] The base sequences of the newly synthesized POR (Cytochrome P450 oxidoreductase) genes from R. norvegicus, B. Taurus, and G. gallus, which were codon-optimized for yeast, are shown in Figures 26a to 26c, respectively. The base sequences of the POR (Cytochrome P450 oxidoreductase) genes from H. sapiens and O. cuniculus are shown in Figures 26d and 26e.

[0549] C.CYB5 (Cytochrome b5) gene

[0550] The base sequences of expression cassettes containing the CYB5 (Cytochrome b5) gene derived from R. norvegicus, O. cuniculus, B. Taurus, and G. gallus, which were newly synthesized by codon optimization in yeast, are as shown in Figures 27a to 27d, respectively. The base sequence of the CYB5 (Cytochrome b5) gene derived from H. sapiens is as shown in Figure 27e.

[0551] 11-3. Cloning primer

[0552] For plasmid construction, the expression cassette containing each gene was amplified using the primers in Table 52 and used to construct the target plasmid.

[0553] Cloning primer direction sequence size (bp) sequence number CAUP_NgoMIV_F forward 5'- gacggggaaagccggcacggattagaagccgccgag -3'36 bp 109 CAUP_SalI_r reverse 5'- tctaatccgtgtcgacaaagccttcgagcgtccc -3'34 bp 110s RnPOR_SacI_F forward 5'- ttaagcttggtaccgagctcaaaaatgggtgattcc -3'36 bp 111s RnPOR_XbaI_r reverse 5'- gatgcggccctctagattaagaccaaacatcc -3'32 bp 112s BtPOR_SacI_F forward 5'- ttaagcttggtaccgagctcaaaaatgaacatgg -3'34 bp113sBtPOR_XbaI_rReverse 5'- gatgcggccctctagattaagaccaaacatccaaag -3'36 bp114sGgPOR_SacI_FForward 5'- ttaagcttggtaccgagctcaaaaatgggtgatgctgg -3'38 bp115sGgPOR_XbaI_rReverse 5'- gatgcggccctctagattaagaccagacatccaaagag -3'38 bp116CYB5_NgoMIV_FForward 5'- gacggggaaagccggcgtcgacccgcggggcgccttaattaaacggattagaagccgccgagc -3'42 bp117CYB5_SpeI_rReverse 5'- taatccgtggactagtaaagccttcgagcgtcccaaaac -3'39 bp118

[0554] 11-4. Plasmid construction through introduction of the CYP8B1 gene

[0555] Expression cassettes containing five novel mammalian CYP8B1 genes were cleaved at NgoMIV and SalI restriction enzyme sites and inserted into pY-sCPBUD(B) and pY-sCPBUD(Hv) plasmids treated with the same restriction enzymes, respectively. The 10 constructed plasmids were named pY-sCPBUDC(BH), pY-sCPBUDC(BR), pY-sCPBUDC(BO), pY-sCPBUDC(BB), pY-sCPBUDC(BG), pY-sCPBUDC(HvH), pY-sCPBUDC(HvR), pY-sCPBUDC(HvO), pY-sCPBUDC(HvB), and pY-sCPBUDC(HvG), and the construction schematic is as shown in Figure 28a.

[0556] 11-5. Construction of plasmids by introducing POR (Cytochrome P450 oxidoreductase) and CYB5 genes

[0557] The introduction of two redox partner enzymes, POR (Cytochrome P450 oxidoreductase) and CYB5 genes, into the pYES2_HIS vector was sequentially constructed. First, the three POR (Cytochrome P450 oxidoreductase) genes were each treated with SacI and XbaI in the pYES2_HIS vector and then constructed. The three plasmids constructed through this method were named pYH-sRnPOR, pYH-sBtPOR, and pYH-sGgPOR, and the construction schematic is as shown in Figure 28b. The construction schematic of pY-sOcPOR, into which the POR (Cytochrome P450 oxidoreductase) gene derived from O. cuniculus was inserted, is as shown in Figure 28c.

[0558] An expression cassette containing a CYB5 gene of the same origin was inserted into a plasmid into which a POR (Cytochrome P450 oxidoreductase) gene was inserted, and the expression cassettes were cut with NgoMIV and SpeI, respectively, to construct plasmids containing two types of redox partner enzymes of the same origin. The four constructed plasmids were named pYH-sOcPH, pYH-sRnPH, pYH-sBtPH, and pYH-sGgPH, respectively, and the construction schematic is as shown in Figure 28d.

[0559] The suitability of the constructed plasmid was determined through plasmid DNA fragmentation. Ten types of plasmids constructed by introducing the CYP8B1 gene were subjected to DNA fragmentation using each restriction enzyme, and the expected fragment sizes upon digestion are shown in Table 53. As shown in Figure 29, the experimental results confirmed that the corresponding sizes were suitable.

[0560] Plasmid nameRestriction enzymeClone sizeExpected sizepYH-sRnPORSacI,XbaI7985 bp2043, 5942 bppYH-sBtPORSacI,XbaI7991 bp2049, 5942 bppYH-sGgPORSacI,XbaI7979 bp2037, 5942 bppYH-sOcPHNgoMIV,SpeI8951 bp1106, 7845 bppYH-sRnPHNgoMIV,SpeI9056 bp1214, 7842 bppYH-sBtPHPstI,SpeI9062 bp2796, 6266 bppYH-sGgPHPstI,SpeI9062 bp2808, 6254 bp

[0561] [Example 12]

[0562] Confirmation of production of 7α,12α-dihydroxycholest-4-en-3-one from various mammalian genetic resources using yeast.

[0563] The production of 7α,12α-dihydroxycholesten-4-en-3-one was confirmed by applying the CYP8B1, a step 3 substance conversion gene from various mammals, to two types of plasmids producing 7α-hydroxy-4-cholesten-3-one (a step 2 substance in the CA biosynthetic pathway).

[0564] The production strain is Saccharomyces cerevisiae BY4741, and the plasmid information is as shown in Table 54.

[0565] Plasmid details pYES2P gal1 E. coliand yeast shuttle vector, 2μ origin,URA3gene, Amp r pY-sCPBUDCpYES2 containing the codon-optimizedCYP7A1, POR(Cytochrome P450 oxidoreductase),CYB5,HSD3B7(transcript variant 2) andCYP8B1genes fromHomo sapiens,andUPC2-1gene fromSaccharomyces cerevisiaepY-sCPBUDC(HvH)pY-sCPBUD(Hv) 1containing the codon-optimizedCYP8B1gene fromH. sapienspY-sCPBUDC(HvR)pY-sCPBUD(Hv) containing the codon-optimizedCYP8B1gene fromRattus norvegicuspY-sCPBUDC(HvO)pY-sCPBUD(Hv) containing the codon-optimizedCYP8B1gene fromOryctolagus cuniculuspY-sCPBUDC(HvB)pY-sCPBUD(Hv) containing the codon-optimizedCYP8B1gene fromBos tauruspY-sCPBUDC(HvG)pY-sCPBUD(Hv) containing the codon-optimizedCYP8B1gene fromGallus galluspY-sCPBUDC(BH)pY-sCPBUD(B) 2 containing the codon-optimizedCYP8B1gene fromH. sapienspY-sCPBUDC(BR)pY-sCPBUD(B) containing the codon-optimizedCYP8B1gene fromR. norvegicuspY-sCPBUDC(BO)pY-sCPBUD(B) containing the codon-optimizedCYP8B1gene fromO. cuniculuspY-sCPBUDC(BB)pY-sCPBUD(B) containing the codon-optimizedCYP8B1gene fromB. tauruspY-sCPBUDC(BG)pY-sCPBUD(B) containing the codon-optimizedCYP8B1gene fromG. galluspYES_HISP Gal1 E. coliand yeast shuttle vector, 2μ origin,HIS3gene, Amp rpYH-sHBpYES2_HIS containing the codon-optimizedPOR(Cytochrome P450 oxidoreductase) andCYB5genes fromH. sapienspYH-sRnPHpYES2_HIS containing the codon-optimizedPOR(Cytochrome P450 oxidoreductase) andCYB5genes fromR. norvegicuspYH-sOcPHpYES2_HIS containing the codon-optimizedPOR(Cytochrome P450 oxidoreductase) andCYB5genes fromO. cuniculuspYH-sBtPHpYES2_HIS containing the codon-optimizedPOR(Cytochrome P450 oxidoreductase) andCYB5genes fromB. tauruspYH-sGgPHpYES2_HIS containing the codon-optimizedPOR(Cytochrome P450 oxidoreductase) andCYB5genes fromG. gallus

[0566] 1 pY-sCPBUD(Hv): pYES2 containing codon-optimized CYP7A1, POR (Cytochrome P450 oxidoreductase), CYB5, and HSD3B7 (full-length form, transcript variant 1) genes from H. sapiens and UPC2-1 gene from S. cerevisiae. 2 pY-sCPBUD(B): pYES2 containing codon-optimized CYP7A1, POR (Cytochrome P450 oxidoreductase) and CYB5 genes from H. sapiens, HSD3B7 genes from B. taurus and UPC2-1 genes from S. cerevisiae

[0567] 12-1. Introduction of expression plasmid into yeast

[0568] The 14 new plasmids and pYH-sHB plasmid constructed in Example 11 were introduced into the BY741 yeast strain in various combinations. The 14 plasmid combinations are shown in Table 55.

[0569] NumberPlasmid IPlasmid IIDetailed information1pY-sCPBUDC(HvH)pYH-sHBCombination of genes of the same species2pY-sCPBUDC(HvR)pYH-sRnPH3pY-sCPBUDC(HvO)pYH-sOcPH4pY-sCPBUDC(HvB)pYH-sBtPH5pY-sCPBUDC(HvG)pYH-sGgPH6pY-sCPBUDC(BH)pYH-sHB7pY-sCPBUDC(BR)pYH-sRnPH8pY-sCPBUDC(BO)pYH-sOcPH9pY-sCPBUDC(BB)pYH-sBtPH10pY-sCPBUDC(BG)pYH-sGgPH11pY-sCPBUDC(HvO)pYH-sHBHeterogeneous genes Combination 12pY-sCPBUDC(HvO)pYH-sRnPH 13pY-sCPBUDC(HvO)pYH-sBtPH 14pY-sCPBUDC(HvO)pYH-sGgPH

[0570] To insert the plasmid into the BY4741 yeast strain with the above combination, Invitrogen's Sc EasyComp TMA transformation kit was used. The transformation method was carried out according to the protocol in the kit (Version C, 110801), and the transformation was performed in the same manner as in Example 3-1. The solid medium for obtaining transformed yeast colonies was SD-His / -Ura agar medium that simultaneously lacks histidine and uracil. To prepare SD-His / -Ura agar medium, 6.7 g of amino acid-free yeast nitrogen base and 0.75 g of -His / -Ura DO (drop out) supplement were dissolved in 800 mL of distilled water, and the pH was adjusted to approximately 5.6 to 6.0. Then, 20 g of agar was added and the final volume was adjusted to 900 mL with distilled water. After autoclaving at 121℃ for 20 minutes and cooling, 100 mL of 20% (w / v) glucose was added, poured into a petri dish, allowed to solidify at room temperature, and then stored in the refrigerator.

[0571] The seed culture medium was the same as the solid medium, SD-His / -Ura medium. 6.7 g of yeast nitrogen base without amino acids and 0.75 g of -His / -Ura DO (drop out) supplement were dissolved in 800 mL of distilled water, the pH was adjusted to approximately 5.6–6.0, and the final volume was made up to 900 mL with distilled water. The medium prepared as above was autoclaved at 121°C for 20 minutes, cooled, and 100 mL of 20% (w / v) glucose was added before storage at room temperature.

[0572] For all strains used in the test, expression was regulated by galactose using the Gal promoter, so galactose was added to the culture medium to induce expression. For the main culture, a complex medium, YPDG medium, was used. To prepare YPDG medium, 10 g of yeast extract and 20 g of peptone were dissolved in 900 mL of distilled water, autoclaved at 121°C for 20 minutes, cooled, and then 50 mL of 20% (w / v) glucose and 50 mL of 20% (w / v) galactose were added. After preparation, it was stored at room temperature.

[0573] Since the test condition was to add cholesterol as a substrate during the main culture, 5% (w / v) cholesterol stock was used and added to the culture medium at a final concentration of 100 mg / L.

[0574] The seed culture was carried out in the same manner as in 1-3. The main culture was carried out in the main culture medium with a final concentration of 0.4 OD. 600nm The seed culture was inoculated to be . After inoculation, it was cultured for 72 hours under the conditions of 30℃ and 180 rpm. The culture was performed in a 60 mL scale in a 250 mL baffled flask, and for each condition, two samples (duplicate) were composed and performed. In this example, three cultures were performed to confirm the homologous gene combination, and two cultures were performed to confirm the heterologous gene combination.

[0575] 12-2. Extraction

[0576] 50 mL of the main culture was centrifuged at 3,000 g for 30 minutes, the supernatant was removed, and 20 mL of 50 mM Tris-HCl (pH 7.5) and approximately 1 to 1.5 g of glass beads were added to the obtained yeast pellet, and vortexed for 5 minutes to disrupt the cells. 5 mL of ethyl acetate was added to separate the layers. To ensure good layer separation, centrifugation was performed at 3,000 g for 10 minutes, and the upper ethyl acetate layer was recovered. This same recovery process was repeated twice using 3 mL of ethyl acetate, and the recovered ethyl acetate supernatant was dried using a rotary vacuum concentrator to prepare a sample for analysis.

[0577] 12-3. Analysis

[0578] To prepare standard solutions, 10 mg each of cholesterol, 7α-hydroxycholesterol, step 2 substances, and step 3 substances were taken, diluted in 10 mL of methanol, and filtered through a 0.22 μm filter. To confirm the homologous combination, a test solution was prepared by concentrating 50 mL of culture broth, adding 1 mL of methanol to the dried sample, diluting it, and centrifuging (12,000 rpm, RT for 10 min). The supernatant was then collected and used for evaluation. A test solution to confirm the heterologous combination was prepared using the same method, but was analyzed using 5 mL of methanol and a final concentration factor of 10.

[0579] The production of each substance was confirmed using the peak area of ​​the test solution obtained by testing the blank test solution (methanol), standard solution, and test solution under the operating conditions of Table 56.

[0580] LC System Column Waters symmetry C18 (4.6 x 75 mm, 3.5 μm) Column temperature 25 ℃ Mobile phase A 100% acetonitrile Mobile phase B 100% methanol Injection volume 10 μL Sample temperature 4 ℃ Run time 20 min Needle wash 80% methanol Seal wash 10% methanol Gradient mode Time (min) Flow (mL / min) A (%) B (%) 0 0.68 0 2 0 8 0 0.68 0 2 0 8 0 1 6 0 1 6 0 1 7 ... Resolution 115.0 Ion Energy 10.8 RM Resolution 22.80 HM Resolution 215.0 Ion Energy 20.5 MRM Mode Compound Cone (V) Collisions (V) Parent Ion (m / z) Daughter Ion (m / z) Cholesterol 2525396.4147.01 Step 1) Step 2525367.5241.02 2) Step 2525401.3383.53 3) 2525417.3269.0

[0581] 1) Step 1: 7α-hydroxycholesterol; 2) Step 2: 7α-hydroxy-4-cholesten-3-one; 3) Step 3: 7α,12α-dihydroxycholest-4-en-3-one

[0582] 12-4. Confirmation of three-stage material production through homologous genetic combinations

[0583] A negative strain containing the empty vectors pYES2 and pYES_HIS and a strain containing the existing 3-step target substance production plasmid pY-sCPBUDC / pYH-sHB were cultured together with a strain containing the plasmids of combinations 1 to 10 of Table 44 above, and a comparison of production volumes was conducted.

[0584] The combinations numbered 1 to 10 in Table 44 were obtained from H. Sapiens, R. norvegicus, O. cuniculus, B. taurus, and G. gallus, and the CYP8B1 gene and the POR (Cytochrome P450 oxidoreductase) and CYB5 genes, which are redox partner enzymes of the same origin, were combined to confirm the production amount of the 3-stage material.

[0585] A total of three cultivations were performed to confirm the production volume and ensure the reproducibility of the test, and the average value was calculated for the results of each strain. As shown in Figure 30a, the OD of the empty vector pYES2 / pYES_HIS 600nm OD of 3-stage material producing strains compared to 600nm I was able to confirm that it was low.

[0586] As a result of confirming the production amount of the 3-step target substance, it was confirmed that approximately 0.031 mg / L of the 3-step target substance was produced in the case of the existing 3-step target substance production plasmid, pY-sCPBUDC / pYH-sHB. Regardless of the origin of the 2-step substance conversion gene (pY-sCPBUD(Hv), pY-sCPBUD(B)), production of the 3-step substance was not confirmed under the conditions in which CYP8B1 from R. norvegicus and B. Taurus was applied (Nos. 2, 4, 7, and 9 in Table 52). Among the remaining 6 combinations in which production was confirmed, the condition with the highest production was pY-sCPBUDC(HvO) / pYH-sOcPH, with an average production amount of approximately 8.5 mg / L over 3 cultures. As confirmed in Table 57, this was the result of an approximately 270-fold increase in the production amount of the 3-step substance compared to the existing conditions.

[0587] Number of strains (mg / L) Cholesterol Stage 1 a Step 2 b Step 3 c 1BY4741 (pYES2 / pYES_HIS)0.741±0.162ND dNDND2BY4741 (pY-sCPBUDC / pYH-sHB)4.764±0.6640.880±0.3320.014±0.0030.031±0.0143BY4741 (pY-sCPBUDC(HvH) / pYH-sHB)5.347±0.7550.048±0.0241.123±0.3013.217±1.1634BY4741 (pY-sCPBUDC(HvR) / pYH-sRnPH)6.286±0.8790.103±0.0092.661±0.336ND5BY4741 (pY-sCPBUDC(HvO) / pYH-sOcPH)5.977±0.9860.032±0.0070.433±0.0738.493±1.5606BY4741 (pY-sCPBUDC(HvB) / pYH-sBtPH)5.376±0.2700.107±0.0142.361±0.378ND7BY4741 (pY-sCPBUDC(HvG) / pYH-sGgPH)6.803±0.9060.056±0.0171.013±0.1294.839±0.8968BY4741 (pY-sCPBUDC(BH) / pYH-sHB)6.379±0.9110.076±0.0201.464±0.0353.959±0.4149BY4741 (pY-sCPBUDC(BR) / pYH-sRnPH)0.631±0.170ND0.004±0.000ND10BY4741 (pY-sCPBUDC(BO) / pYH-sOcPH)5.999±1.2080.043±0.0130.547±0.0517.420±0.69511BY4741 (pY-sCPBUDC(BB) / pYH-sBtPH)6.115±0.8920.139±0.0293.129±0.737ND12BY4741 (pY-sCPBUDC(BG) / pYH-sGgPH)5.052±0.9140.047±0.0150.454±0.1112.515±0.739

[0588] a 7α-hydroxycholesterol; b 7α-hydroxy-4-cholesten-3-one; c 7α,12α-hydroxycholest-4-en-3-one; d Not detected

[0589] 12-5. Confirmation of three-stage material production through heterologous gene combinations

[0590] As a result of confirming the production through the combination of homologous genes, the production was the highest in the combination derived from O. cuniculus, and among them, the production was the highest with the pY-sCPBUDC(HvO) / pYH-sOcPH plasmid. Therefore, we tried to confirm the possibility of increasing the production through the combination of pY-sCPBUDC(HvO) and a heterologous redox partner enzyme gene. As in the homologous gene combination test, the strains containing the empty vectors pYES2 and pYES_HIS were used as negative control strains, and the production was compared using the existing 3-step target substance production conditions, pY-sCPBUDC / pYH-sHB, and the homologous gene combination pY-sCPBUDC(HvO) / pYH-sOcPH as positive control strains.

[0591] A total of two cultivations were performed to confirm the production volume and ensure the reproducibility of the test. As shown in Figure 30b, the average value for the results of each strain was calculated, and the OD of the empty vector pYES2 / pYES_HIS 600nm OD of 3-stage material producing strains compared to 600nm It was confirmed that OD was low. Among them, the condition of the pY-sCPBUDC(HvO) / pYH-sRnPH combination was low in the latter half of the culture. 600nm It was confirmed that the temperature was lowered.

[0592] As a result of the culture, the production amount of pY-sCPBUDC(HvO) / pYH-sOcPH was confirmed to be approximately 8.9 mg / L, which was similar to the 8.5 mg / L confirmed when searching for homologous genes. Among the strains in which heterologous genes were combined, the combination that showed a higher production amount than the pY-sCPBUDC(HvO) / pYH-sOcPH condition was pY-sCPBUDC(HvO) / pYH-sRnPH, with a production amount of approximately 13.2 mg / L, which was an approximately 1.5-fold improvement compared to the homologous gene combination. In this test, an absolute comparison is impossible because the 3-stage production amount of the existing strain was not confirmed during sample analysis, but in a relative comparison, as confirmed in Table 58, it can be said that the 3-stage material production amount was improved by approximately 400 times compared to the existing condition.

[0593] Number of strains (mg / L) Cholesterol Stage 1 a Step 2 b Step 3 c 1BY4741 (pYES2 / pYES_HIS)0.970±0.192ND d NDND2BY4741 (pY-sCPBUDC / pYH-sHB)6.235±0.2510.538±0.025NDND3BY4741 (pY-sCPBUDC(HvO) / pYH-sOcPH)7.560±0.103ND0.333±0.0598.938±1.1384BY4741 (pY-sCPBUDC(HvO) / pYH-sHB)6.413±0.547ND0.235±0.0276.568±0.591.5BY4741 (pY-sCPBUDC(HvO) / pYH-sRnPH)7.280±0.718ND0.655±0.06713.218±1.4216BY4741 (pY-sCPBUDC(HvO) / pYH-sBtPH)7.740±0.614ND0.225±0.0178.798±0.9707BY4741 (pY-sCPBUDC(HvO) / pYH-sGgPH)8.005±0.215ND0.260±0.0128.933±0.412

[0594] a7α-hydroxycholesterol; b 7α-hydroxy-4-cholesten-3-one; c 7α,12α-hydroxycholest-4-en-3-one; d Not detected

[0595] Based on the above results, the 3-step material conversion gene was selected from O. cuniculus, and two types, pYH-sOcPH plasmid using the homologous gene and pYH-sRnPH using the heterologous gene, were selected and used as subsequent step material conversion gene resources.

[0596] [Example 13]

[0597] Construction of a plasmid producing 7α,12α-dihydroxy-5β-cholestan-3-one within a novel CA pathway from diverse genetic resources.

[0598] In order to construct a CA synthetic pathway in yeast, genes of various origins involved in the conversion of cholesterol to 7α,12α-dihydroxy-5β-cholestan-3-one (CAS No. 547-97-7, hereinafter referred to as a step 4 product) were inserted into a yeast expression vector to construct a plasmid producing the step 4 product of the CA pathway.

[0599] 13-1. Template vector

[0600] Step 4 conversion genes of the CA pathway of various origins were inserted into the pYH-sOcPH and pYH-sRnPH plasmids.

[0601] 13-2. Insert gene

[0602] The base sequences of expression cassettes containing the AKR1D1 (Δ4-3-oxosteroid 5β-reductase, EC No. 1.3.1.3) gene derived from Homo sapiens (transcript variant 2 type (H) and transcript variant 1 type (Hv)), Bos taurus, Sus scrofa, Oryctolagus cuniculus, Rattus norvegicus, Gallus gallus, Xenopus laevis, and Fusarium verticillioides, which were newly synthesized by codon optimization in yeast, are as shown in Figures 31a to 31i, respectively.

[0603] 13-3. Cloning primer

[0604] For plasmid construction, the expression cassettes containing each gene were amplified using the primers in Table 56 and used to construct the target plasmid.

[0605] Cloning primer direction sequence size (bp) sequence number CAUP NF forward 5'-gctggcgccacggattagaagccgccgagc-3'30bp 119 CAUP PR reverse 5'-cgattaattaaagccttcgagcgtccc-3'27bp 120

[0606] 13-4. Construction of a 4-step material production plasmid using the pYH-sOcPH plasmid

[0607] Expression cassettes containing nine new AKR1D1 genes of various origins were cleaved at the NarI and PacI restriction enzyme sites and inserted into the pYH-sOcPH plasmids treated with the same restriction enzymes. The nine constructed plasmids were named pYH-sOcPHD(H), pYH-sOcPHD(Hv), pYH-sOcPHD(B), pYH-sOcPHD(S), pYH-sOcPHD(O), pYH-sOcPHD(R), pYH-sOcPHD(G), pYH-sOcPHD(X), and pYH-sOcPHD(F), respectively, and the construction schematic is as shown in Figure 32a.

[0608] 13-5. Construction of a 4-step material production plasmid using the pYH-sRnPH plasmid.

[0609] Among the nine new AKR1D1 genes of various origins, expression cassettes including those of H. sapiens (transcript variant 1 type (Hv)), B. taurus, and G. gallus were each inserted into the pYH-sRnPH plasmid using the same method as above. The three additionally constructed plasmids were named pYH-sRnPHD (Hv), pYH-sRnPHD (B), and pYH-sRnPHD (G), respectively, and the construction schematic is as shown in Figure 32b.

[0610] To determine whether the plasmid was properly constructed, plasmid DNA fragmentation was performed. Twelve plasmids constructed by introducing the AKR1D1 gene were subjected to DNA fragmentation using each restriction enzyme, and the expected fragment sizes upon digestion are shown in Table 60. As shown in Figure 33, the experimental results confirmed that the sizes were appropriate.

[0611] Plasmid nameRestriction enzymeClone sizeExpected sizepYH-sOcPHD(H)HpaI10584 bp2387, 8197 bppYH-sOcPHD(Hv)HpaI10707 bp2510, 8197 bppYH-sOcPHD(B)HpaI10707 bp2510, 8197 bppYH-sOcPHD(S)EcoRI10707 bp2271, 8436 bppYH-sOcPHD(O)EcoRI10707 bp2271, 8436 bppYH-sOcPHD(R)HpaI10707 bp2510, 8197 bppYH-sOcPHD(G)EcoRI10707 bp2384, 8323 bppYH-sOcPHD(X)EcoRI10341 bp2384, 7957 bppYH-sOcPHD(F)NdeI10662 bp3801, 6861 bppYH-sRnPHD(Hv)SacI10812 bp1190, 1764, 7858 bppYH-sRnPHD(B)SacI10812 bp1190, 1764, 7858 bppYH-sRnPHD(G)SacI10812 bp1190, 1764, 7858 bp

[0612] AKR1D1, a step 4 conversion gene of the CA pathway, was selected from various genetic resources and a total of 12 plasmids producing step 4 materials were constructed.

[0613] [Example 14]

[0614] Confirmation of production of 7α,12α-dihydroxy-5β-cholestan-3-one (step 4) from various genetic resources using yeast.

[0615] A total of eight 7α,12α-dihydroxy-5β-cholestan-3-one (the target substance of the step 4) conversion gene AKR1D1 were applied to two types of plasmids producing 7α,12α-dihydroxycholest-4-en-3-one (the target substance of the step 3 in the CA biosynthetic pathway) to confirm the production amount of the target substance of the step 4.

[0616] The production strain is Saccharomyces cerevisiae BY4741, and the plasmid information is as shown in Table 61.

[0617] Plasmid details pYES2P gal1 E. coliand yeast shuttle vector, 2μ origin,URA3gene, Amp r pY-sCPBUDCpYES2 containing the codon-optimizedCYP7A1, POR(Cytochrome P450 oxidoreductase),CYB5,HSD3B7(transcript variant 2) andCYP8B1genes fromHomo sapiens,andUPC2-1gene fromS. cerevisiaepY-sCPBUDC(HvO)pY-sCPBUD(Hv) 1) containing the codon-optimizedCYP8B1gene fromOryctolagus cuniculuspYES_HISP Gal1 E. coliand yeast shuttle vector, 2μ origin,HIS3gene, Amp r pYH-sHBDpYH-sHB 2) containing the codon-optimizedAKR1D1gene(transcript variant 2) fromH. sapienspYH-sOcPHD(H)pYH-sOcPHD 3)containing the codon-optimizedAKR1D1gene(transcript variant 2) fromH. sapienspYH-sOcPHD(Hv)pYH-sOcPH containing the codon-optimizedAKR1D1gene(Full form, transcript variant 1) fromH. sapienspYH-sOcPHD(B)pYH-sOcPH containing the codon-optimizedAKR1D1gene fromBos tauruspYH-sOcPHD(S)pYH-sOcPH containing the codon-optimizedAKR1D1gene fromSus scrofapYH-sOcPHD(O)pYH-sOcPH containing the codon-optimizedAKR1D1gene fromO. cuniculuspYH-sOcPHD(R)pYH-sOcPH containing the codon-optimizedAKR1D1gene fromRattus norvegicuspYH-sOcPHD(G)pYH-sOcPH containing the codon-optimizedAKR1D1gene fromGallus galluspYH-sOcPHD(X)pYH-sOcPH containing the codon-optimizedAKR1D1gene fromXenopus laevispYH-sOcPHD(F)pYH-sOcPH containing the codon-optimizedAKR1D1gene fromFusarium verticillioidespYH-sRnPHD(Hv)pYH-sRnPH 4)containing the codon-optimizedAKR1D1gene(Full form, transcript variant 1) fromH. sapienspYH-sRnPHD(B)pYH-sRnPH containing the codon-optimizedAKR1D1gene fromB. tauruspYH-sRnPHD(G)pYH-sRnPH containing the codon-optimizedAKR1D1gene fromG. gallus

[0618] 1) pY-sCPBUD(Hv): pYES2 containing codon-optimized CYP7A1, POR (Cytochrome P450 oxidoreductase), CYB5, and HSD3B7 (full-length form, transcript variant 1) genes from H. sapiens and UPC2-1 gene from S. cerevisiae. 2) pYH-sHB: pYES2_HIS containing codon-optimized POR (Cytochrome P450 oxidoreductase) and CYB5 genes from H. sapiens

[0619] 3) pYH-sOcPH: pYES2_HIS containing codon-optimized POR (Cytochrome P450 oxidoreductase) and CYB5 genes from O. cuniculus

[0620] 4) pYH-sRnPH:pYES2_HIS containing codon-optimized POR (Cytochrome P450 oxidoreductase) and CYB5 genes from R. norvegicus

[0621] 14-1. Introduction of expression plasmid into yeast

[0622] In order to introduce the 12 new plasmids constructed in Example 13 into yeast, Sc EasyComp from Invitrogen was used. TMA transformation kit was used. The transformation method was carried out according to the protocol in the kit (Version C, 110801), and transformation was performed in the same manner as in Example 3-1.

[0623] In this example, a three-step material production strain (BY4741 (pY-sCPBUDC (HvO)) was produced as a soluble cell. Transformation was performed by mixing the prepared soluble cells and plasmid, adding solution III, and reacting in a 30°C water bath for 1 hour.

[0624] Information on each yeast strain used in the test and abbreviations to be written are as shown in Table 62.

[0625] 번호플라스미드 I플라스미드 II정보약어1pYESpYES_HISNegative controlY / YH2pY-sCPBUDC(HvO)pYH-sOcPHStep 3 controlHvO / OcPH3pY-sCPBUDCpYH-sHBDStep 4 controlH / HBD4pY-sCPBUDC(HvO)pYH-sOcPHD(H)AKR1D1fromH. sapiens(w / O. cRedox partner enzyme)HvO / OcPHD(H)5pY-sCPBUDC(HvO)pYH-sOcPHD(Hv)AKR1D1(v1)fromH. sapiens(w / O. cRedox partner enzyme)HvO / OcPHD(Hv)6pY-sCPBUDC(HvO)pYH-sOcPHD(B)AKR1D1fromB. taurus(w / O. cRedox partner enzyme)HvO / OcPHD(B)7pY-sCPBUDC(HvO)pYH-sOcPHD(S)AKR1D1fromS. scrofa(w / O. cRedox partner enzyme)HvO / OcPHD(S)8pY-sCPBUDC(HvO)pYH-sOcPHD(O)AKR1D1fromO. cuniculus(w / O. cRedox partner enzyme)HvO / OcPHD(O)9pY-sCPBUDC(HvO)pYH-sOcPHD(R)AKR1D1fromR. norvegicus(w / O. cRedox partner enzyme)HvO / OcPHD(R)10pY-sCPBUDC(HvO)pYH-sOcPHD(G)AKR1D1fromG. gallus(w / O. cRedox partner enzyme)HvO / OcPHD(G)11pY-sCPBUDC(HvO)pYH-sOcPHD(X)AKR1D1fromX. laevis(w / O. cRedox partner enzyme)HvO / OcPHD(X)12pY-sCPBUDC(HvO)pYH-sOcPHD(F)AKR1D1fromF. verticillioides(w / O.cRedox partner enzyme)HvO / OcPHD(F)13pY-sCPBUDC(HvO)pYH-sRnPHD(Hv)AKR1D1(v1)fromH. sapiens(w / R. nRedox partner enzyme)HvO / RnPHD(Hv)14pY-sCPBUDC(HvO)pYH-sRnPHD(B)AKR1D1fromB. taurus(w / R. nRedox partner enzyme)HvO / RnPHD(B)15pY-sCPBUDC(HvO)pYH-sRnPHD(G)AKR1D1fromG. gallus(w / R.nRedox partner enzyme)HvO / RnPHD(G).

[0626] Media and reagents were prepared and used in the same manner as in Example 12-1, and seed and main cultures were also conducted in the same manner as in Example 12-1. At this time, cholesterol was added to the culture medium to a final concentration of 100 mg / L. To compare the production of each strain, cultures, extractions, and analyses were performed at least twice.

[0627] 14-2. Extraction

[0628] The analysis sample was prepared by performing extraction in the same manner as in Example 12-2.

[0629] 14-3. Analysis

[0630] To prepare standard solutions, 10 mg each of cholesterol, 7α-hydroxycholesterol, and step 2, step 3, and step 4 substances were taken, diluted in 10 mL of methanol, and filtered through a 0.22 μm filter. The test solution was prepared by adding 1 mL of methanol to the concentrated and dried sample extracted from 50 mL of culture medium, diluting it, and centrifuging it (12,000 rpm, RT conditions, 10 minutes).

[0631] The production of each substance was confirmed using the peak area of ​​the test solution obtained by testing the blank test solution (methanol), standard solution, and test solution under the operating conditions of Table 56. At this time, the MRM mode is as shown in Table 63.

[0632] MRM ModeCompoundCon (V)Collision (V)Mother Ion (m / z)Daughter Ion (m / z)Cholesterol2525396.4147.01 Step 1) Step 2525367.5241.02 2) Step 2525401.3383.53 3) Step 2525417.3269.04 4) 2520401.2383.2

[0633] 1) Step 1: 7α-hydroxycholesterol 2) Step 2: 7α-hydroxy-4-cholesten-3-one

[0634] 3) Step 3: 7α,12α-dihydroxycholest-4-en-3-one

[0635] 4) Step 4: 7α,12α-dihydroxy-5β-cholestan-3-one

[0636] 14-4. Confirmation of production of target substance in 4 steps using pYH-sOcPH plasmid

[0637] Using the selected 3-step target substance production plasmid pYH-sOcPH, the 4-step substance conversion gene AKR1D1 of various origins (H. sapiens (transcript 1, 2), B. Taurus, S. scrofa, O. cuniculus, R. norvegicus, G. gallus, X. laevis, F. verticillioides) was applied, and a total of 9 strains using the new plasmid were constructed. The constructed new strains are as shown in numbers 4 to 12 in Table 51, and as controls for these strains, a 3-step target substance production strain (number 2 in Table 51) and a 4-step target substance production strain (number 3 in Table 51) containing all of the 1-4 step conversion genes derived from H. sapiens (transcript 2) were used and cultured in a complex medium.

[0638] As a result of confirming the production amount of each step substance of the strain produced through two cultures, the step 4 substance was not detected in the step 3 substance producing strain HvO / OcPH and the step 4 substance producing strains H / HBD, HvO / OcPHD(H), HvO / OcPHD(X), and HvO / OcPHD(F). In the remaining 6 strains using the HvO / OcPHD(Hv), HvO / OcPHD(B), HvO / OcPHD(S), HvO / OcPHD(O), HvO / OcPHD(R), and HvO / OcPHD(G) plasmids, the step 4 target substance was detected, and among these, in the case of HvO / OcPHD(S), HvO / OcPHD(O), and HvO / OcPHD(R), the production amount of the step 4 substance was extremely small, and it was confirmed that the step 3 substance, which is the precursor, was accumulated. In the case of HvO / OcPHD(Hv), HvO / OcPHD(B), and HvO / OcPHD(G), the production amount of precursor materials for stages 1 to 3 was low, while the production amount of material for stage 4 was high. This confirmed that materials for stages 1 to 3 were efficiently converted into target materials for stage 4 when using the three genetic resources. Based on these results, two additional cultivations were performed for HvO / OcPHD(Hv), HvO / OcPHD(B), and HvO / OcPHD(G), where the conversion of target materials was well achieved. Finally, pYH-sOcPH OD for the culture results of 9 new strains and 2 control strains using plasmids 600nm The average and standard deviation of the production amount of the target material at each stage were calculated (Figure 34a and Table 64).

[0639] Number of strains (abbreviation) Concentration (mg / L) Cholesterol Stage 1 a Step 2 b Step 3 c Step 4 d 1HvO / OcPH6.987±1.7330.016±0.0020.259±0.0519.811±2.002ND e2H / HBD10.258±2.1120.748±0.1530.008±0.0010.059±0.016ND3HvO / OcPHD(H)10.137±0.1700. 021±0.0030.279±0.01710.668±0.835ND4HvO / OcPHD(Hv)8.494±1.6140.021±0.0030.171±0.06 00.797±0.1618.768±0.8355HvO / OcPHD(B)7.903±1.6530.016±0.0010.115±0.0180.271±0.056 8.386±0.8566HvO / OcPHD(S)7.647±1.5780.019±0.0060.215±0.0568.119±1.0970.392±0.0897H vO / OcPHD(O)6.492±3.3060.018±0.0020.223±0.0408.909±1.6530.318±0.0148HvO / OcPHD(R)8 .674±0.3880.016±0.0010.251±0.0189.222±1.3310.002±0.0009HvO / OcPHD(G)7.800±2.0000.0 14±0.0040.144±0.0400.320±0.1007.855±1.54110HvO / OcPHD(X)8.007±0.6590.015±0.0010.2 08±0.0158.350±0.560ND11HvO / OcPHD(F)8.628±1.4200.016±0.0020.237±0.0438.763±1.637ND

[0640] a 7α-hydroxycholesterol; b 7α-hydroxy-4-cholesten-3-one; c 7α,12α-dihydroxycholest-4-en-3-one; d 7α,12α-dihydroxy-5β-cholestan-3-one; e Not detected

[0641] 14-5. Confirmation of production of target substance in step 4 using pYH-sRnPH plasmid

[0642] In order to confirm the production of the 4th step target substance, pYH-RnPH, which had a high production rate of the 3rd step substance in a combination of redox partner enzymes of different origins from the above, was used. To this end, three types of plasmids were additionally secured by applying genes derived from H. sapiens (transcript 1), B. Taurus, and G. gallus, which had high production rates of the 4th step substance among the above results. The results of the additionally secured HvO / RnPHD(Hv), HvO / RnPHD(B), and HvO / RnPHD(G) (Nos. 13, 14, and 15 in Table 51) were cultured twice to compare them with those of HvO / OcPHD(Hv), HvO / OcPHD(B), and HvO / OcPHD(G) (Nos. 5, 6, and 10 in Table 51).

[0643] As a result, we were able to confirm the production of 4-stage substances of about 6 mg / L or more under all conditions, and pYH-sOcPH The conditions using plasmids showed a tendency for higher production, although slightly different from those using pYH-sRnPH plasmids. Culture OD using seven types of plasmids, including the negative control group 600nm The average and standard deviation of the production amount of the target material at each stage are as shown in Figure 34b and Table 65.

[0644] Number of strains (abbreviation) Concentration (mg / L) Cholesterol Stage 1 a Step 2 b Step 3 c Step 4 d 1YES / YES_HIS0.432±0.073ND eNDNDND2HvO / OcPHD(Hv)3.992±0.548ND0.444±0.2750.707±0.0616.865±0.8213HvO / OcPHD(B)4.080±0.564 ND0.225±0.1050.305±0.0477.054±1.0184HvO / OcPHD(G)4.273±0.537ND0.288±0.1070.416±0.0576.869±1. 0405HvO / RnPHD(Hv)4.456±0.211ND0.455±0.2220.671±0.0816.362±0.2536HvO / RnPHD(B)4.220±0.453ND0 .178±0.0320.205±0.0305.964±0.6517HvO / RnPHD(G)4.429±0.727ND0.288±0.0680.333±0.0616.122±0.622

[0645] a 7α-hydroxycholesterol; b 7α-hydroxy-4-cholesten-3-one; c 7α,12α-dihydroxycholest-4-en-3-one; d 7α,12α-dihydroxy-5β-cholestan-3-one; e Not detected

[0646] Among the previously developed CA production pathways, the combinations of pY-sCPBUDC(HvO) / pYH-sOcPH and pY-sCPBUDC(HvO) / pYH-sRnPH plasmids were selected as a result of the verification of production of materials in steps 1 to 3 and the application of various derived genes of conversion genes. Based on these results, a total of nine types of the four-step target material conversion gene AKR1D1 were applied to the pYH-sOcPH and pYH-sRnPH plasmids. A total of 12 types of four-step target material production plasmids were constructed, and yeast strains were constructed, and the production of the four-step target material and the production amount were confirmed by culturing them.

[0647] A total of 9 new plasmids using pYH-sOcPH were produced, and the step 4 target substance was detected in 6 of them. Among them, 3 types, HvO / OcPHD(Hv), HvO / OcPHD(B), and HvO / OcPHD(G), showed high production of the step 4 substance at about 8 mg / L. In addition, the amounts of the step 1-3 target substances were confirmed to be very small in the above 3 types. Considering that the production amount of the step 3 target substance, which is a precursor, was about 9 mg / L, it can be said that most of the produced step 3 substances were converted to step 4 substances. Finally, among the step 4 production plasmids using pYH-sOcPH, the condition with the highest production amount was the condition using a gene derived from H. sapiens (transcript 1), and the step 4 target substance production was confirmed to be about 8.77 mg / L.

[0648] In the preliminary test, another plasmid selected when the production of the 3-step target substance was confirmed, pYH-sRnPH, showed a 3-step target substance production rate that was approximately 1.5 times higher than that of the pYH-sOcPH plasmid condition (approximately 8.5 mg / L using pYH-sOcPH, approximately 13.2 mg / L using pYH-sRnPH). By applying genes derived from H. sapiens (transcript 1), B. Taurus, and G. gallus, which had high 4-step target substance production rates, to pYH-sRnPH, we aimed to compare the 4-step production rates with those under the conditions using pYH-sOcPH. As a result of the test, no significant increase in production was confirmed between the conditions using pYH-sOcPH and pYH-sRnPH, and contrary to the results of the 3-step production confirmation, the conditions using the pYH-sOcPH plasmid showed a relatively small but high tendency compared to the conditions using pYH-sRnPH.

[0649] Based on the above results, pYH-sOcPHD(Hv) and pYH-sOcPHD(B) were selected using the pYH-sOcPH plasmid, which applied the 4-step conversion genes from H. Sapiens (transcript variant 1) and B. Taurus.

[0650] [Example 15]

[0651] Construction of a plasmid for producing 3α,7α,12α-trihydroxy-5β-cholestane (step 5) in a novel CA pathway from various genetic resources.

[0652] In order to construct a CA synthetic pathway in yeast, genes of various origins involved in the conversion of cholesterol to 3α,7α,12α-trihydroxy-5β-cholestane (CAS No. 547-96-6, hereinafter referred to as the 5-step product) were inserted into a yeast expression vector to construct a plasmid producing the 5-step product of the CA pathway.

[0653] 15-1. Template vector

[0654] The five step conversion genes of the CA pathway, synthesized from various sources, were inserted into three CA-producing four-step plasmids, pYH-sOcPHD(Hv), pYH-sOcPHD(B), and pYH-sRnPHD(B).

[0655] 15-2. Insert gene

[0656] The base sequences of expression cassettes containing the AKR1C4 gene derived from Homo sapiens, Macaca fascicularis, Saimiri boliviensis boliviensis, Nomascus leucogenys, Gorilla gorilla gorilla and the hsdA gene derived from Comamonas testosteroni, which were newly synthesized by codon optimization in yeast, are as shown in Figures 35a to 35f, respectively.

[0657] 15-3. Cloning primer

[0658] For plasmid construction, the expression cassettes containing each gene were amplified using the primers in Table 66 and used to construct the target plasmid.

[0659] Cloning primer direction Sequence size (bp) Sequence number CAUP SF forward 5'-gctccgcggacggattagaagccgccgagc-3'30121 CAUP NR reverse 5'-gttttgggacgctcgaaggctttggcgcctcg-3'32122

[0660] 15-4. Construction of a 5-step material production plasmid using the pYH-sOcPHD(Hv) plasmid.

[0661] Expression cassettes containing six AKR1C4 genes synthesized from various sources were cleaved at SacII and NarI restriction enzyme sites and inserted into the pYH-sOcPHD(Hv) plasmid treated with the same restriction enzymes. The six constructed plasmids were named pYH-sOcPHDC(HvH), pYH-sOcPHDC(HvM), pYH-sOcPHDC(HvSb), pYH-sOcPHDC(HvN), pYH-sOcPHDC(HvGg), and pYH-sOcPHDC(HvC), respectively, and the construction schematic is as shown in Figure 36a.

[0662] 15-5. Construction of a 5-step material production plasmid using the pYH-sOcPHD(B) plasmid.

[0663] Using the pYH-sOcPHD(B) plasmid, six AKR1C4 genes of various origins were inserted in the same manner as above. The six constructed plasmids were named pYH-sOcPHDC(BH), pYH-sOcPHDC(BM), pYH-sOcPHDC(BSb), pYH-sOcPHDC(BN), pYH-sOcPHDC(BGg), and pYH-sOcPHDC(BC), respectively, and the construction schematic is as shown in Figure 36b.

[0664] 15-6. Construction of a 5-step material production plasmid using the pYH-sRnPHD(B) plasmid.

[0665] Using the pYH-RnPHD(B) plasmid, hsdA from C. testosteroni was inserted in the same manner as described above. One of the constructed plasmids was named pYH-sRnPHDC(BC), and the construction schematic is shown in Figure 36c.

[0666] To determine whether the plasmid was properly constructed, it was determined through plasmid DNA fragmentation. 13 types of plasmids constructed by introducing the AKR1C4 and hsdA genes were subjected to DNA fragmentation using each restriction enzyme, and the expected fragment sizes upon digestion are shown in Table 67. As shown in Figures 37a to 37c, it was confirmed that the experimental results were suitable for the corresponding sizes.

[0667] Plasmid nameRestriction enzymeClone sizeExpected sizepYH-sOcPHDC(HvH)HpaI12456 bp4259, 8197 bppYH-sOcPHDC(HvM)PstI12456 bp4540, 7916 bppYH-sOcPHDC(HvSb)PstI12540 bp2716, 4540, 5284 bppYH-sOcPHDC(HvN)PstI12453 bp4540, 7913 bppYH-sOcPHDC(HvGg)PstI12456 bp4540, 4916 bppYH-sOcPHDC(HvC)PstI12258 bp4540, 7718 bppYH-sOcPHDC(BH)EcoRI12456 bp3905, 8551 bppYH-sOcPHDC(BM)PstI12456 bp4540, 7916 bppYH-sOcPHDC(BSb)EcoRI12540 bp3905, 8635 bppYH-sOcPHDC(BN)EcoRI12453 bp3905, 8548 bppYH-sOcPHDC(BGg)EcoRI12456 bp3905, 8551 bppYH-sOcPHDC(BC)PstI12258 bp4540, 7718 bppYH-sRnPHDC(BC)SacII,SpeI12363 bp7857, 4506 bp

[0668] AKR1C4, a step 5 conversion gene of the CA pathway, was selected from various genetic resources and a total of 13 plasmids producing step 5 materials were constructed.

[0669] [Example 16]

[0670] Confirmation of production of 3α,7α,12α-trihydroxy-5β-cholestane (step 5) from various genetic sources using yeast.

[0671] Two types of plasmids producing 7α, 12α-dihydroxy-5β-cholestan-3-one (the target substance of step 4 in the CA biosynthetic pathway) were selected, and genes of various origins involved in the conversion of 3α, 7α, 12α-trihydroxy-5β-cholestane (hereinafter referred to as the step 5 product) were applied to confirm the production amount of the step 5 target substance.

[0672] The production strain is Saccharomyces cerevisiae BY4741, and the plasmid information is as shown in Table 68.

[0673] Plasmid details pYES2P gal1 E. coliand yeast shuttle vector, 2μ origin,URA3gene, Amp r pY-sCPBUDC(HvO)pY-sCPBUD(Hv) 1 containing the codon-optimizedCYP8B1gene fromOryctolagus cuniculuspYES_HISP Gal1 E. coliand yeast shuttle vector, 2μ origin,HIS3gene, Amp r pYH-sOcPHD(HvH)pYH-sOcPHD(Hv) 1)containing the codon-optimizedAKR1C4gene fromHomo sapienspYH-sOcPHDC(HvM)pYH-sOcPHD(Hv) containing the codon-optimizedAKR1C4 gene from Macaca fascicularispYH-sOcPHDC(HvSb)pYH-sOcPHD(Hv) containing the codon-optimizedAKR1C4 gene from Saimiri boliviensis boliviensispYH-sOcPHDC(HvN)pYH-sOcPHD(Hv) containing the codon-optimizedAKR1C4 gene from Nomascus leucogenyspYH-sOcPHDC(HvGg)pYH-sOcPHD(Hv) containing the codon-optimizedAKR1C4 gene from Gorilla gorilla gorillapYH-sOcPHDC(HvC)pYH-sOcPHD(Hv) containing the codon-optimizedhsdA gene from Comamonas testosteronipYH-sOcPHDC(BH)pYH-sOcPHD(B) 2)containing the codon-optimizedAKR1C4gene fromH. sapienspYH-sOcPHDC(BM)pYH-sOcPHD(B) containing the codon-optimizedAKR1C4 gene from M. fascicularispYH-sOcPHDC(BSb)pYH-sOcPHD(B) containing the codon-optimizedAKR1C4 gene from Sb boliviensispYH-sOcPHDC(BN)pYH-sOcPHD(B) containing the codon-optimizedAKR1C4 gene from N.leucogenyspYH-sOcPHDC(BGg)pYH-sOcPHD(B) containing the codon-optimizedAKR1C4 gene from G. g. gorillapYH-sOcPHDC(BC)pYH-sOcPHD(B) containing the codon-optimizedhsdA gene from C. testosteronipYH-sRnPHDC(BC)pYH-sRnPHD(B) 3) containing the codon-optimizedhsdA gene from C. testosteroni

[0674] 1) pYH-sOcPHD(Hv): pYES2_HIS containing codon-optimized POR (Cytochrome P450 oxidoreductase) and CYB5 genes from O. cuniculus, and AKR1D1 gene (transcript variant 1) from H. sapiens 2) pYH-sOcPHD(B): Codon-optimized POR (Cytochrome P450 oxidoreductase) and CYB5 genes from O. cuniculus, and Bos taurus origin Containing the AKR1D1 gene pYES2_HIS

[0675] 3)pYH-sRnPHD(B): Codon-optimized POR (Cytochrome P450 oxidoreductase) and CYB5 genes from Rattus norvegicus, and B. taurus pYES2_HIS containing the AKR1D1 gene

[0676] 16-1. Introduction of expression plasmid into yeast

[0677] In order to introduce 13 new plasmids constructed using the pYH-sOcPHD(Hv) and pYH-sOcPHD(B) plasmids in Example 15 into yeast, Sc EasyComp from Invitrogen was used. TM A transformation kit was used. The transformation method was carried out according to the protocol in the kit (Version C, 110801), and transformation was performed in the same manner as in Example 4-1.

[0678] In this example, a three-step material production strain (BY4741 (pY-sCPBUDC (HvO)) was produced as a soluble cell. Transformation was performed by mixing the prepared soluble cells and plasmid, adding solution III, and reacting in a 30°C water bath for 1 hour.

[0679] Information on each yeast strain used in the test and abbreviations to be written are as shown in Table 69.

[0680] 번호플라스미드 I플라스미드 II정보약어1pY-sCPBUDC(HvO)pYH-sOcPHDC(HvH)AKR1D1(v1)andAKR1C4fromH. sapiens(w / O.cRedox partner enzyme)HvO / OcPHDC(HvH)2pY-sCPBUDC(HvO)pYH-sOcPHDC(HvM)AKR1D1(v1)fromH. sapiensandAKR1C4fromM.a fascicularis(w / O.cRedox partner enzyme)HvO / OcPHDC(HvM)3pY-sCPBUDCpYH-sOcPHDC(HvSb)AKR1D1(v1)fromH. sapiensandAKR1C4fromS.b. boliviensis(w / O.cRedox partner enzyme)HvO / OcPHDC(HvSb)4pY-sCPBUDC(HvO)pYH-sOcPHDC(HvN)AKR1D1(v1)fromH. sapiensandAKR1C4fromN.leucogenys(w / O.cRedox partner enzyme)HvO / OcPHDC(HvN)5pY-sCPBUDC(HvO)pYH-sOcPHDC(HvGg)AKR1D1(v1)fromH. sapiensandAKR1C4fromG. g. gorilla(w / O.cRedox partner enzyme)HvO / OcPHDC(HvGg)6pY-sCPBUDC(HvO)pYH-sOcPHDC(HvC)AKR1D1(v1)fromH. sapiensandhsdAfromC. testosteroni(w / O.cRedox partner enzyme)HvO / OcPHDC(HvC)7pY-sCPBUDC(HvO)pYH-sOcPHDC(BH)AKR1D1fromB.taurusandAKR1C4fromH. sapiens(w / O.cRedox partner enzyme)HvO / OcPHDC(BH)8pY-sCPBUDC(HvO)pYH-sOcPHDC(BM)AKR1D1fromB.taurusandAKR1C4fromM.a fascicularis(w / O.cRedox partner enzyme)HvO / OcPHDC(BM)9pY-sCPBUDC(HvO)pYH-sOcPHDC(BSb)AKR1D1fromB.taurusandAKR1C4fromS.b. boliviensis(w / O.cRedox partner enzyme)HvO / OcPHDC(BSb)10pY-sCPBUDC(HvO)pYH-sOcPHDC(BN)AKR1D1fromB.taurusandAKR1C4fromN.leucogenys(w / O.cRedox partner enzyme)HvO / OcPHDC(BN)11pY-sCPBUDC(HvO)pYH-sOcPHDC(BGg)AKR1D1fromB.taurusandAKR1C4fromG. g. gorilla(w / O.cRedox partner enzyme))HvO / OcPHDC(BGg)12pY-sCPBUDC(HvO)pYH-sOcPHDC(BC)AKR1D1fromB.taurusandhsdAfromC. testosteroni(w / O.cRedox partner enzyme)HvO / OcPHDC(BC)13pY-sCPBUDC(HvO)pYH-sRnPHDC(BC)AKR1D1fromB.taurusandhsdAfromC. testosteroni(w / R.nRedox partner enzyme)HvO / RnPHDC(BC).

[0681] Media and reagents were prepared and used in the same manner as in Example 12-1, and seed and main cultures were also conducted in the same manner as in Example 12-1. At this time, cholesterol was added to the culture medium to a final concentration of 100 mg / L. To compare the production of each strain, cultures, extractions, and analyses were performed at least twice.

[0682] 16-2. Extraction

[0683] The analysis sample was prepared by performing extraction in the same manner as in Example 12-2.

[0684] 16-3. Analysis

[0685] To prepare standard solutions, 10 mg each of cholesterol, 7α-hydroxycholesterol, and step 2, step 3, step 4, and step 5 substances were taken, diluted in 10 mL of methanol, and filtered through a 0.22 μm filter. The test solution was prepared by adding 1 mL of methanol to the concentrated and dried sample extracted from 50 mL of culture medium, diluting it, and centrifuging it (12,000 rpm, RT conditions, 10 minutes).

[0686] The production of each substance was confirmed using the peak area of ​​the test solution obtained by testing the blank test solution (methanol), standard solution, and test solution under the operating conditions of Table 56. At this time, the MRM mode is as shown in Table 70.

[0687] MRM ModeCompoundCon (V)Collision (V)Mother Ion (m / z)Daughter Ion (m / z)Cholesterol2525396.4147.01 Step 1) Step 2525367.5241.02 2) Step 2525401.3383.53 3) Step 2525417.3269.04 4) Step 2520401.2383.25 5) 2520385.2367.2

[0688] 1) Step 1: 7α-hydroxycholesterol; 2) Step 2: 7α-hydroxy-4-cholesten-3-one; 3) Step 3: 7α,12α-dihydroxycholest-4-en-3-one; 4) Step 4: 7α,12α-dihydroxy-5β-cholestan-3-one; 5) Step 5: 3α,7α,12α-trihydroxy-5β-cholestane

[0689] 16-4. Confirmation of production of target substances in 5 steps using YH-sOcPHD(Hv) and pYH-sOcPHD(B) plasmids

[0690] Using the selected 4-step target substance production plasmids pYH-sOcPHD(Hv) and pYH-sOcPHD(B), 12 new strains were constructed by applying AKR1C4 of various origins (H. sapiens, M. fascicularis, Sbboliviensis, N. leucogenys, G. g. gorilla) and hsdA gene of C. testosteroni, respectively. The constructed new strains are as numbered 1 to 12 in Table 71, and a total of 12 strains were cultured in a complex medium up to 3 times to determine the OD 600nm The average and standard deviation of the production amount of the target material at each stage were calculated (Fig. 38a, Table 71).

[0691] Number Strain (Abbreviation) Concentration (mg / L) Cho a Step 1 b Step 2 c Step 3 d Step 4 e Step 5 f 1HvO / OcPHDC(HvH)6.653±2.976ND g1.610±1.9863.126±1.0353.659±0.5770.231±0.0322HvO / OcPHDC(HvM)6.898±2.870ND0.922±0.9662.752±0.9293.437±0.922 3.362±0.9923HvO / OcPHDC(HvSb)3.078±1.924ND0.599±0.8591.070±0.6473.39±2.7110.116±0.1574HvO / OcPHDC(HvN)6.250± 1.997ND1.623±1.9783.999±0.6965.880±2.5410.253±0.0695HvO / OcPHDC(HvGg)6.555±2.504ND1.819±2.2534.868±0.5624.7 35±1.6620.495±0.1386HvO / OcPHDC(HvC)6.207±1.747ND1.564±2.0563.739±1.3760.409±0.0874.738±1.1117HvO / OcPHDC(BH )7.388±2.367ND0.368±0.1890.602±0.0398.887±3.3250.448±0.0988HvO / OcPHDC(BM)6.891±2.675ND0.306±0.1700.469±0.1 075.247±2.0283.520±1.3529HvO / OcPHDC(BSb)6.765±2.054ND0.445±0.2800.811±0.0949.492±3.6480.068±0.01510HvO / OcP HDC(BN)10.402±0.252ND0.244±0.0120.809±0.03513.02±0.7390.306±0.00811HvO / OcPHDC(BGg)10.399±0.293ND0.217±0.00 70.550±0.01612.22±0.4760.916±0.07012HvO / OcPHDC(BC)6.111±1.821ND0.115±0.0560.047±0.0090.190±0.0346.118±1.676

[0692] a cholesterol; b 7α-hydroxycholesterol; c 7α-hydroxy-4-cholesten-3-one; d7α,12α-dihydroxycholest-4-en-3-one; e 7α,12α-dihydroxy-5β-cholestan-3-one; f 3α,7α,12α-trihydroxy-5β-cholestane; g Not detected

[0693] As a result, production of the 5-step target substance was confirmed under all conditions, but the production amount showed differences depending on the gene origin. In the case of the conditions using AKR1C4 derived from H. sapiens, S. boliviensis, N. leucogenys, and G. g. gorilla, the production amount of the 5-step target substance was confirmed to be very low at less than 1 mg / L in all conditions (HvO / OcPHDC(HvH), HvO / OcPHDC(HvSb), HvO / OcPHDC(HvN), HvO / OcPHDC(HvGg), HvO / OcPHDC(BH), HvO / OcPHDC(BSb), HvO / OcPHDC(BN), HvO / OcPHDC(BGg)) using pYH-sOcPHD(Hv) and pYH-sOcPHD(B). In contrast, AKR1C4 derived from M. fascicularis and C. In the case of HvO / OcPHDC(HvM), HvO / OcPHDC(HvC), HvO / OcPHDC(BM), and HvO / OcPHDC(BC), which are conditions using hsdA derived from testosteroni, the production amounts of target substances in the five stages were confirmed to be approximately 3.362 mg / L, 4.738 mg / L, 3.520 mg / L, and 6.118 mg / L, respectively, in the order presented above.

[0694] 16-5. Confirmation of production of target substance in 5 steps using pYH-sRnPHD(B) plasmid

[0695] A new plasmid was constructed by introducing hsdA from C. testosteroni, which had the highest production of material in the 5th stage, into pYH-sRnPHD(B) (plasmid for producing material in the 4th stage). This was cultured under the same conditions as those using the pYH-sOcPHD(Hv) and pYH-sOcPHD(B) plasmids to compare the production of material in the 5th stage. OD was calculated for a total of 11 conditions, excluding the condition using the plasmid using the gene from S. boliviensis, which had the lowest production in the 5th stage (No. 3 and 9 in Table 71). 600nm The average and standard deviation of the production amount of each target substance at each stage were calculated (Fig. 38b, Table 72).

[0696] Number of strains (mg / L)Cho a Step 1 b Step 2 c Step 3 d Step 4 e Step 5 f 1HvO / OcPHDC(HvH)6.422±0.418ND g0.168±0.0002.271±0.2296.678±0.1920.325±0.0192HvO / OcPHDC(HvM)6.417±0.249ND0.156±0.0081.262±0.1923.319 ±0.2493.270±0.0323HvO / OcPHDC(HvN)7.065±0.055ND0.2215±0.0094.055±0.2178.250±0.1000.275±0.0034HvO / OcPH DC(HvGg)6.136±0.088ND0.73±0.0092.956±0.0706.002±0.1300.537±0.0035HvO / OcPHDC(HvC)6.068±0.310ND0.127±0 .0012.238±0.5020.334±0.0364.580±0.2526HvO / OcPHDC(BH)7.485±0.645ND0.174±0.0020.389±0.04510.370±0.2060 .406±0.0447HvO / OcPHDC(BM)7.011±0.039ND0.146±0.0080.282±0.0245.786±0.4043.481±0.0878HvO / OcPHDC(BN)7.7 11±0.139ND0.203±0.0070.595±0.02311.306±0.0180.279±0.0159HvO / OcPHDC(BGg)7.169±0.1210.024±0.0000.163±0 .0030.382±0.0329.752±0.1660.703±0.02310HvO / OcPHDC(BC)6.593±0.4010.008±0.0000.116±0.0020.039±0.0010.2 15±0.0117.043±0.06511HvO / RnPHDC(BC)8.059±0.3000.021±0.0090.177±0.0090.040±0.0010.258±0.0117.978±0.401

[0697] a cholesterol; b 7α-hydroxycholesterol; c 7α-hydroxy-4-cholesten-3-one; d 7α,12α-dihydroxycholest-4-en-3-one; e 7α,12α-dihydroxy-5β-cholestan-3-one;f 3α,7α,12α-trihydroxy-5β-cholestane; g Not detected

[0698] As a result of this test, the same tendency was observed when confirming the production amount for each gene as in the first test, and among the genes used, the conditions using genes derived from M. fascicularis and C. testosteroni had the highest production amount of the 5-stage substance. In addition, it was confirmed that the production amount of the 5-stage substance was higher under the condition using the gene derived from C. testosteroni than under the condition using the gene derived from M. fascicularis. Finally, the production amount of the 5-stage substance under the three conditions using hsdA derived from C. testosteroni was confirmed to be approximately 4.580 mg / L for HvO / OcPHDC (HvC), approximately 7.043 mg / L for HvO / OcPHDC (BC), and approximately 7.978 mg / L for HvO / RnPHDC (BC).

[0699] Based on the results of the application of various derived genes of the conversion genes and the confirmation of the production of materials for steps 1 to 4 in the CA production pathway, the plasmids producing the 4-step materials were selected as the combinations of pY-sCPBUDC(HvO) / pYH-sOcPHD(Hv), pY-sCPBUDC(HvO) / pYH-sOcPHD(B), and pY-sCPBUDC(HvO) / pYH-sRnPHD(B). Based on these results, among the plasmids producing the three 4-step materials, a total of six types of 5-step target material conversion genes, AKR1C4 or hsdA, were applied to pYH-sOcPHD(Hv), pYH-sOcPHD(B), and pYH-sRnPHD(B), to create a final 13 types of 5-step target material production plasmids. The manufactured plasmid was introduced into yeast to create a strain producing the 5-step target substance, and this strain was cultured to confirm whether the 5-step target substance was produced and the amount produced.

[0700] As a result of introducing six new 5-step material production plasmids into two plasmids, pYH-sOcPHD(Hv) and pYH-sOcPHD(B), for each of 12 conditions, production of 5-step materials was confirmed under all conditions. In addition, when the production was compared by culturing under each of the six conditions using pYH-sOcPHD(Hv) and pYH-sOcPHD(B), it was confirmed that the production tended to be similar depending on each introduced gene. H. sapiens, Sbboliviensis, N. leucogenys, G. g. The conditions using AKR1C4 from gorilla, HvO / OcPHDC(HvH), HvO / OcPHDC(HvSb), HvO / OcPHDC(HvN), HvO / OcPHDC(HvGg), HvO / OcPHDC(BH), HvO / OcPHDC(BSb), HvO / OcPHDC(BN), HvO / OcPHDC(BGg), showed low production of the 5-step target substance at less than 1 mg / L, while the conditions using AKR1C4 from M. fascicularis and hsdA from C. testosteroni, HvO / OcPHDC(HvM), HvO / OcPHDC(HvC), HvO / OcPHDC(BM), HvO / OcPHDC(BC), showed high production of the 5-step target substance at more than 3 mg / L. However, M. When using genes derived from M. fascicularis and C. testosteroni, the production of the 4th stage substance was confirmed to be at a similar level to the 5th stage substance under the conditions using AKR1C4 derived from M. fascicularis (HvO / OcPHDC(HvM), HvO / OcPHDC(BM)), and the production of the 4th stage substance was confirmed to be low under the conditions using hsdA derived from C. testosteroni (HvO / OcPHDC(HvC), HvO / OcPHDC(BC)), confirming that the conversion to the 5th stage substance was carried out efficiently.As a result, the production of the 5-stage material was the highest under the HvO / OcPHDC(BC) condition, and the confirmed production of the 5-stage material was approximately 6.118 mg / L.

[0701] Based on these results, hsdA from C. testosteroni was introduced into another 4-step material production plasmid pYH-sRnPHD(B) to construct a plasmid, and the HvO / RnPHDC(BC) strain was obtained through yeast strain construction. This was co-cultured with conditions where 5-step material production was confirmed, and the target material production amount was compared. Ultimately, the 5-step material production was the highest under the HvO / RnPHDC(BC) condition, and the confirmed 5-step material production amount was approximately 7.978 mg / L.

[0702] Based on the above results, the hsdA gene derived from C. testosteroni was selected as a 5-step target substance conversion gene.

[0703] [Example 17]

[0704] Construction of a plasmid producing 7α-hydroxy-5β-cholestan-3-one within a novel CDCA pathway from genetic resources of various origins.

[0705] In order to construct a CDCA synthetic pathway in yeast, genes of various origins involved in the conversion of cholesterol to 7α-hydroxy-5β-cholestan-3-one (hereinafter, CDCA pathway step 3 product) were inserted into a yeast expression vector to construct a plasmid producing a step 3 product of the CDCA pathway.

[0706] 17-1. Template vector

[0707] The three-step conversion genes of the CDCA pathway of various origins were inserted into the pYES2_HIS vector.

[0708] 17-2. Insert gene

[0709] The base sequences of the newly synthesized AKR1D1 (Δ4-3-oxosteroid 5β-reductase, 1.3.1.3) genes derived from Homo sapiens, Bos taurus, Sus scrofa, Oryctolagus cuniculus, Rattus norvegicus, Gallus gallus, Xenopus laevis, and Fusarium verticillioides by codon optimization in yeast are as shown in Figures 39a to 39h, respectively. In the case of the AKR1D1 gene derived from H. sapiens, the gene used in the existing production was transcript variant 2 type, but a new full-length form (transcript variant 1) was additionally introduced. The sequence of the existing AKR1D1 gene is as shown in Figure 39i.

[0710] 17-3. Cloning primer

[0711] For plasmid construction, the expression cassettes containing each gene were amplified using the primers in Table 73 and used to construct the target plasmid.

[0712] Cloning primer direction sequence size (bp) sequence number s HsAKR1D1_SacI_F forward 5'- ttaagcttggtaccgagctcaaaaatggatttgtc -3'35 bp 123 s HsAKR1D1_XbaI_r reverse 5'- gatgcggccctctagattagtattcatc -3'28 bp 124 s BtAKR1D1_SacI_F forward 5'- ttaagcttggtaccgagctcaaaaatgcacttgtc -3'35 bp 125 s SsAKR1D1_SacI_F forward 5'- ttaagcttggtaccgagctcaaaaatgtacttgtc -3'35 bp 126 s Rn AKR1D1_SacI_F forward 5'- ttaagcttggtaccgagctcaaaaatgaacttgtc -3'35 bp127sGgAKR1D1_SacI_Fforward5'- taagcttggtaccgagctcaaaaatgtctttgacc -3'35 bp128sXlAKR1D1_SacI_Fforward5'- ttaagcttggtaccgagctcaaaaatggcttttaagcc -3'38 bp129sFvAKR1D1_XbaI_rReverse5'- gatgcggccctctagattaccaaacttgc -3'29 bp130

[0713] 17-4. Plasmid construction

[0714] Nine new AKR1D1 genes of various origins were cleaved with SacI and XbaI restriction enzyme sites and inserted into the pYES2_HIS vector treated with the same restriction enzymes. The nine constructed plasmids were named pYH-sHsAKR1D1, pYH-sHsAKR1D1(v1), pYH-sBtAKR1D1, pYH-sSsAKR1D1, pYH-sOcAKR1D1, pYH-sRnAKR1D1, pYH-sGgAKR1D1, pYH-sXlAKR1D1, and pYH-sFvAKR1D1, respectively, and the construction schematic is as shown in Figure 40.

[0715] To determine whether the construct was constructed, plasmid DNA fragmentation and DNA sequencing were performed. The results of the plasmid DNA fragmentation were as follows.

[0716] Nine types of plasmids constructed by introducing the AKR1D1 gene were fragmented using their respective restriction enzymes, and the expected fragment sizes upon digestion are shown in Table 74. As shown in Figure 41, the experimental results confirmed that the sizes were appropriate.

[0717] Plasmid nameRestriction enzymeClone sizeExpected sizepYH-sHsAKR1D1EcoRV,PstI6806 bp2770, 4036 bppYH-sHsAKR1D1(v1)EcoRV,PstI6929 bp2893, 4036 bppYH-sBtAKR1D1ScaI6929 bp1827, 5102 bppYH-sSsAKR1D1ScaI6929 bp1827, 5102 bppYH-sOcAKR1D1ScaI6929 bp1827, 5102 bppYH-sRnAKR1D1ScaI6929 bp1827, 5102 bppYH-sGgAKR1D1ScaI6929 bp1827, 5102 bppYH-sXlAKR1D1ScaI6563 bp1827, 4736 bppYH-sFvAKR1D1NdeI6884 bp2956, 3928 bp

[0718] [Example 18]

[0719] Construction of a plasmid producing 3α,7α-dihydroxy-5β-cholestanate within the CDCA pathway

[0720] To construct a CDCA synthetic pathway in yeast, a production candidate plasmid was constructed using Homo sapiens-derived genes for the conversion of cholesterol to 3α,7α-dihydroxy-5β-cholestanate (hereinafter referred to as the 7th step product of the CDCA pathway).

[0721] 18-1. Template vector

[0722] The three-step material production plasmids pYH-sHsAKR1D1(v1) and pYH-sBtAKR1D1 were used to introduce the four-step and seven-step material production genes and three redox partner enzymes of the seven-step material conversion enzyme.

[0723] 18-2. Insert gene

[0724] The 4-step material conversion gene is AKR1C4 (3α-hydroxysteroid 3-dehydrogenase (Si-specific), 1.1.1.50), and a gene derived from H. sapiens was used. When introduced into the 3-step material production plasmid, it was used in the form of the pY-sHsAKR1C4 plasmid inserted into the pYES2 vector using the SacI and XbaI restriction enzyme sites. The nucleotide sequence of the corresponding expression cassette is as shown in Figure 42a.

[0725] The step 7 material conversion gene is CYP27A1 (cholestanetriol 26-monooxygenase, 1.14.15.15). The gene is a cytochrome P450 enzyme, and its functional expression requires co-expression with the redox partner enzyme ferredoxin reductase (FDXR), so a candidate gene was selected. The gene selected as the redox partner enzyme is FDXR derived from H. sapiens. Each gene was used in the form of pY-sHsCYP27A1 and pY-sHsFDXR plasmids inserted into the pYES2 vector using SacI and XbaI restriction enzyme sites. The nucleotide sequences of the corresponding expression cassettes are as shown in Figures 42b and 42c.

[0726] 18-3. Cloning primer

[0727] To construct six types of plasmids, the primers in Table 75 were used to amplify the expression cassettes containing each gene, and used to construct the target plasmids.

[0728] Cloning primer direction sequence size (bp) sequence number CAUP NSSNP-F forward 5'-GACGGGGAAAGCCGGCGTCGACCCGCGGGGCGCCTTAATTAAACGGATTAGAAGCCGCCGAG -3'62 bp 131CAUP SR reverse 5'-TAATCCGTGGACTAGTAAAGCCTTCGAGCGTCCC -3'35 bp 132CAUP NF forward 5'-TCGACCCGCGGGGCGCCACGGATTAGAAGCCGCCGAG -3'37 bp 133CAUP PR reverse 5'-CTTCTAATCCGTTTAATTAAAGCCTTCGAGCGTCCC -3'36 bp 134CAUP SF forward 5'-AGCCGGCGTCGACCCGCGGACGGATTAGAAGCCGCCG - 3'37 bp 135CAUP NRreverse 5'-TTCTAATCCGTGGCGCCAAAGCCTTCGAGCGTCCC -3'35 bp136

[0729] 18-4. Plasmid construction

[0730] The genes for the four-step convertase, AKR1C4 and CYP27A1, were sequentially inserted into the three-step material production plasmids, pYH-sHsAKR1D1(v1) and pYH-sBtAKR1D1. As shown in Figure 43a, each was amplified together with the Gal promoter expression cassette and introduced by digestion with NgoMIV and SpeI restriction enzymes. The constructed plasmids were named pYH-sHsDC(HvH) and pYH-sBtDC(H), respectively.

[0731] The CYP27A1 gene, a 7-step converting enzyme, was inserted into the 4-step material production plasmids constructed above, pYH-sHsDC(HvH) and pYH-sBtDC(H). As shown in Figure 43b, each was amplified together with a Gal promoter expression cassette and introduced by digestion with NarI and PacI restriction enzymes. The constructed plasmids were named pYH-sHsDCC(HvHH) and pYH-sBtDCC(HH), respectively.

[0732] The FDXR gene derived from H. sapiens, which was selected as a redox partner enzyme to be expressed together with the CYP27A1 gene, a 7-step material conversion enzyme, was also amplified together with the Gal promoter expression cassette as shown in Figure 43c and introduced by digestion with SacII and NarI restriction enzymes. The constructed plasmids were named pYH-sHsDCChF (HvHH) and pYH-sBtDCChF (HH), respectively.

[0733] To determine whether the plasmid was properly constructed, plasmid DNA fragmentation and DNA sequencing were performed. The results of the plasmid DNA fragmentation are as follows.

[0734] When the pYH-sHsDC(HvH) and pYH-sBtDC(H) plasmids were fragmented using restriction enzymes PstI and SacI, respectively, the expected fragment sizes were 2646 and 5921 bp and 1757 and 6810 bp, respectively. As shown in Fig. 44a, it was confirmed that the two newly constructed plasmids were both suitable for the corresponding sizes in the experimental results.

[0735] When the pYH-sHsDCC(HvHH) and pYH-sBtDCC(HH) plasmids were fragmented using the restriction enzyme NdeI, the expected fragment sizes for both were 4253 and 6685 bp, respectively. As shown in Fig. 44b, it was confirmed that both newly constructed plasmids were suitable for the corresponding sizes in the experimental results.

[0736] When the pYH-sHsDCChF (HvHH) and pYH-sBtDCChF (HH) plasmids were fragmented using the restriction enzyme HpaI, the expected fragment sizes for both were 4292 and 9028 bp, respectively. As shown in Fig. 44c, it was confirmed that both newly constructed plasmids were suitable for the corresponding sizes in the experimental results.

[0737] [Example 19]

[0738] Confirmation of production of CDCA biosynthetic pathway 7α-hydroxy-5β-cholestan-3-one (step 3), 3α,7α-dihydroxy-5β-cholestan (step 4), and 3α,7α-dihydroxy-5β-cholestanide (step 7) using yeast.

[0739] A total of eight expression plasmids were constructed using five genes involved in the conversion of 7α-hydroxy-4-cholesten-3-one (CAS: 3862-25-7), a step 2 substance of the CA pathway, to a step 7 substance of the CDCA pathway. The production amounts of the target substances of the CDCA biosynthetic pathway, 7α-hydroxy-5β-cholesten-3-one (step 3), 3α,7α-dihydroxy-5β-cholesten (step 4), and 3α,7α-dihydroxy-5β-cholestanate (step 7), were confirmed.

[0740] The production strain is Saccharomyces cerevisiae BY4741, and the plasmid information is as shown in Table 76.

[0741] Plasmid Details Remarks pY-sCPBUD(Hv)pYES2 containing the codon-optimizedCYP7A1, POR(Cytochrome P450 oxidoreductase),CYB5andHSD3B7(v1) genes fromHomo sapiens, andUPC2-1gene fromSaccharomyces cerevisiaeCA and CDCA pathway 2 steps pYH-sHsAKR1D1(v1)pYES2_HIS containing the codon-optimizedAKR1D1(v1) genes fromH. sapiensCDCA pathway 3 steps pYH-sBtAKR1DpYES2_HIS containing the codon-optimizedAKR1D1genes fromBos tauruspYH-sHsDC(HvH)pYH-sHsAKR1D1(v1) containing the codon-optimizedAKR1C4genes fromH. sapiensCDCA step 4 pYH-sBtDC(H)pYH-sBtAKR1D1 containing the codon-optimizedAKR1C4genes fromH. sapienspYH-sHsDCC(HvHH)pYH-sHsDC(HvH) containing the codon-optimized CYP27A1 genes from H. sapiensCDCA step 7 pYH-sBtDCC(HH)pYH-sBtDC(H) containing the codon-optimizedCYP27A1genes fromH. sapienspYH-sHsDCChF(HvHH)pYH-sHsDCC(HvHH) containing the codon-optimizedFDXRgenes fromH. sapienspYH-sBtDCChF(HH)pYH-sBtDCC(HH) containing the codon-optimizedFDXRgenes fromH. sapiens

[0742] 19-1. expression plasmid yeast

[0743] Invitrogen's Sc EasyComp was used to introduce eight newly constructed novel plasmids into yeast. TM A transformation kit was used. The transformation method was carried out according to the protocol in the kit (Version C, 110801), and transformation was performed in the same manner as in Example 4-1.

[0744] In this example, a three-step material production strain (BY4741 (pY-sCPBUD (Hv)) was produced as a soluble cell. Transformation was performed by mixing the prepared soluble cells with the plasmid presented in Table 76, adding Solution III, and reacting in a 30°C water bath for 1 hour.

[0745] Media and reagents were prepared and used in the same manner as in Example 12-1, and seed and main cultures were also conducted in the same manner as in Example 12-1. At this time, cholesterol was added to the culture medium to a final concentration of 100 mg / L. To compare the production of each strain, cultures, extractions, and analyses were performed at least twice.

[0746] 19-2. Extraction

[0747] The analysis sample was prepared by performing extraction in the same manner as in Example 12-2.

[0748] 19-3. Analysis

[0749] To prepare standard solutions, 10 mg each of CA and CDCA pathway step 2, CDCA pathway step 3, step 4, and step 5 materials were taken, diluted in 10 mL of methanol, and filtered through a 0.22 μm filter. The test solution was prepared by adding 1 mL of methanol to the concentrated and dried sample extracted from 50 mL of culture medium, diluting it, and centrifuging it (12,000 rpm, RT conditions, 10 min).

[0750] The production of each substance was confirmed using the peak area of ​​the test solution obtained by testing the blank test solution (methanol), standard solution, and test solution under the operating conditions shown in Table 77.

[0751] LC System Column Waters symmetry C18 (4.6 x 75 mm, 3.5 mm) Column Temperature 25°C Mobile Phase A 100% Methanol Mobile Phase B 100% Acetonitrile Injection Volume 10 mL Sample Temperature 4°C Run Time 20 min Needle Wash 80% Methanol Seal Wash 10% Methanol Gradient Mode Time (min) Flow (mL / min) A (%) B (%) 0 0.6 2 0 8 0 8 0 8 5 1 0 8 0 2 0 1 6 5 1 0 8 0 2 0 1 7 0 6 2 0 8 0 2 0 0.6 2 0 8 0 MS System Ionization Source APCI Corona 4.0 mA Cone 25 V Capillary 4.0 kVA PCI Probe Temperature 350°C N2 Gas Desolvation Flow 400 L / hr RM Resolution 13.0 HM Resolution 115.0 Ion energy 10.8 RM Resolution 23.0 HM Resolution 215.0 Ion energy 20.5 MRM Mode Compound Parent ion (m / z) Daughter ion (m / z) Cholesterol 369.4 147.0 1 step 1) Step 367.5241.02 2) 401.3383.5CDCA-3 Step 3) 385.3231.2CDCA-4 Step 4) 369.5161.2CDCA-7 Step 5) 399.5257.2

[0752] 1) Step 1: 7α-hydroxycholesterol; 2) Step 2: 7α-hydroxy-4-cholesten-3-one; 3) CDCA Step 3: 7α-hydroxy-5β-cholestan-3-one; 4) CDCA Step 4: 3α,7α-dihydroxy-5β-cholestane; 5) CDCA Step 7: 3α,7α-dihydroxy-5β-cholestanate

[0753] 19-4. Confirmation of production of target substances in CDCA stages 3, 4, and 7

[0754] Based on the results of CA 4-step target substance production, the AKR1D1 gene from H. sapiens and B. taurus was selected, and the CDCA 4-step AKR1C4 gene, the 7-step CYP27A1 gene, and the auxiliary enzyme FDXR were introduced from H. sapiens into the CDCA 3-step target substance production plasmids pYH-sHsAKR1D1(v1) and pYH-sBtAKR1D, respectively, to construct a total of 9 strains. The strains were cultured in the prepared complex medium and the OD 600nm And the production amount of each stage target material was calculated (Fig. 45, Table 78).

[0755] Number of strains (mg / L)Cho 1) CA / CDCA Stage 1 2) CA / CDCA Stage 2 3) CDCA3 stage 4) CDCA4 stage 5) CDCA7 stage 6) 1BY4741(pY-sCPBUD(Hv))9.830.051.34ND 7)NDND2BY4741(pY-sCPBUD(Hv) / pYH-sHsAKR1D1(Hv))10.03ND1.010.16N.DND3BY4741(pY-sCPBUD(Hv) / pYH-sBtAKR1D1)8.75ND0.440.40N.DND4BY 4741(pY-sCPBUD(Hv) / pYH-sHsDC(HvH))9.760.031.010.090.01ND5BY4741(pY-sCPBUD(Hv) / pYH-sBtDC(H))9.680.030.760.360.03ND6BY4741(pY -sCPBUD(Hv) / pYH-sHsDCC(HvHH))13.800.030.700.05N.D.0.517BY4741(pY-sCPBUD(Hv) / pYH-sBtDCC(HH))11.800.030.390.170.020.378BY4741 (pY-sCPBUD(Hv) / pYH-sHsDCChF(HvHH))11.120.030.860.05N.D.0.239BY4741(pY-sCPBUD(Hv) / pYH-sBtDCChF(HH))11.470.030.740.210.010.21

[0756] 1) cholesterol; 2) 7α-hydroxycholesterol; 3) 7α-hydroxy-4-cholesten-3-one; 4) 7α-hydroxy-5β-cholestan-3-one; 5) 3α,7α-dihydroxy-5β-cholestane; 6) 3α,7α-dihydroxy-5β-cholestanate; 7) Not detected

[0757] Based on these results, it was confirmed that CDCA stage 3, 4, and 7 substances were also produced.

[0758] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0759] file: / E: / 3DP / D%EB%93%9C%EB%9D%BC%EC%9D%B4%EB%B8%8C%EC%9E%90%EB%A3%8C / WORK / %EC%95%84%EC%9B%83%EA%B3%A0%EC%9E%89%EC%B6%9C%EC%9B%90 / %EB %8C%80%EC%9B%85%EC%A0%9C%EC%95%BD / %EC%9A%B0%EB%A5%B4%EC%86%8C%EB%8 D%B0%EC%98%A5%EC%8B%9C%EC%BD%9C%EB%A6%B0%EC%82%B0 / 1020240046979.xml

Claims

A genetically modified cell producing an ursodeoxycholic acid (UDCA) precursor comprising at least one gene encoding an enzyme involved in a metabolic pathway that synthesizes an ursodeoxycholic acid (UDCA) precursor from cholesterol, The above enzyme is, i) An enzyme that converts cholesterol into 7α-hydroxycholesterol; ii) An enzyme that converts 7α-hydroxycholesterol to 7α-hydroxycholest-4-en-3-one; iii) An enzyme that converts 7α-Hydroxycholest-4-en-3-one to 4-cholesten-7α,12α-diol-3-one; iv) An enzyme that converts 4-cholesten-7α,12α-diol-3-one to 7α,12α-dihydroxy-5β-cholestan-3-one; v) An enzyme that converts 7α,12α-dihydroxy-5β-cholestan-3-one to 3α,7α,12α-trihydroxy-5β-cholestane; vi) An enzyme that converts 7α-Hydroxycholest-4-en-3-one to 7α-Hydroxy-5β-cholestan-3-one; vii) an enzyme that converts 7α-hydroxy-5β-cholestan-3-one to 3α,7α-dihydroxy-5β-cholestane; and viii) An enzyme that converts 3α,7α-dihydroxy-5β-cholestane to 3α,7α-dihydroxy-5β-cholestane via 3α,7α,26-trihydroxy-5β-cholestane and 3α,7α-dihydroxy-5β-cholestane-26-al, and then to 3α,7α-dihydroxy-5β-cholestanate. The genes encoding the above enzymes are operably linked in sequence according to the metabolic pathway that synthesizes the UDCA precursor. In the first claim, the UDCA precursor is 7α-hydroxycholesterol, 7α-hydroxycholest-4-en-3-one, 4-cholesten-7α,12α-diol-3-one, 7α,12α-dihydroxy-5β-cholestane-3-one, 3α,7α,12α-trihydroxy-5β-cholestane, 3α,7α,12α,26-tetrahydroxy-5β-cholestane, 3α,7α,12α-trihydroxy-5β-cholestane-26-al, 3α,7α,12α-trihydroxy-5β-cholestanoate, (25R)-3α,7α,12α-trihydroxy-5β-cholestane-26-oil-CoA, (25S)-3α,7α,12α-trihydroxy-5β-cholestane-26-oleoyl-CoA, 3α,7α,12α-trihydroxy-5β-cholestane-24-enoyl-CoA, 3α,7α,12α,24-tetrahydroxy-5β-24-cholestanoyl-CoA, 3α,7α,12α,trihydroxy-5β-24-oxocholestanoyl-CoA, choleyl-CoA, 7α-hydroxy-5β-cholestane-3-one, 3α,7α-dihydroxy-5β-cholestane, 3α,7α,26-trihydroxy-5β-cholestane, 3α,7α-dihydroxy-5β-cholestane-26-al, 3α,7α-dihydroxy-5β-cholestanate, (25R)-3α,7α-dihydroxy-5β-cholestanoyl-CoA, (25S)-3α,7α-dihydroxy-5β-cholestanoyl-CoA, (24E)-3α,7α-dihydroxy-5β-cholest-24-enoyl-CoA, 3α,7α,24-trihydroxy-5β-cholestanoyl-CoA, 3α,7α-dihydroxy-5β-24-oxocholestanoyl-CoA, chenodeoxycholoyl-CoA, cholic acid (CA) or chenodeoxycholic acid (CDCA). In the second paragraph, a cell wherein the enzyme involved in the CA metabolic pathway among the enzymes is at least one enzyme selected from the group consisting of i) to v). In the second paragraph, a cell wherein the enzyme involved in the CDCA metabolic pathway among the enzymes is at least one enzyme selected from the group consisting of i), ii), and vi) to viii). In the first paragraph, the gene encoding the enzyme of i) is a cell derived from human (Homo sapiens) or rabbit (Oryctolagus cuniculus). In claim 1, the gene encoding the enzyme of ii) is ERG26 derived from yeast (Saccharomyces cerevisiae), hsdD derived from Mycobacterium tuberculosis H37Rv, choD derived from Mycobacterium tuberculosis H37Rv, choB derived from Brevibacterium sterolicum, YKL107W derived from yeast (Saccharomyces cerevisiae), 3BHSD derived from Levilactobacillus brevis, transcript variant 1 of full-length form HSD3B7 derived from human (Homo sapiens), HSD3B7 derived from rabbit (Oryctolagus cuniculus), or HSD3B7 derived from cow (Bos Taurus). In the first paragraph, the gene encoding the enzyme of iii) is a cell derived from human (Homo sapiens), rabbit (Oryctolagus cuniculus) or chicken (Gallus gallus). In the first paragraph, a cell wherein the gene encoding the enzyme of iv) is a transcript variant 1 of human (Homo sapiens) derived AKR1D1, bovine (Bos Taurus) derived AKR1D1, or chicken (Gallus gallus) derived AKR1D1. In the first paragraph, the gene encoding the enzyme of v) is AKR1C4 derived from monkey (Macaca fascicularis) or hsdA derived from Comamonas testosteroni. In the first paragraph, the gene encoding the enzyme of vi) is a cell which is a transcript variant 1 of human (Homo sapiens) derived AKR1D1, bovine (Bos Taurus) derived AKR1D1, or chicken (Gallus gallus) derived AKR1D1. In the first paragraph, the genes encoding the enzyme of vii) and the enzyme of viii) are human (Homo sapiens) derived AKR1C4 and human (Homo sapiens) derived CYP27A1, respectively. A cell according to claim 1, further comprising a UPC2-1 gene. In claim 11, the UPC2-1 gene is a cell derived from yeast. A cell in claim 5, further comprising a gene encoding the enzyme of i) and a gene encoding cytochrome p450 oxidoreductase (POR) and cytochrome b5 (CYB5) of homologous or heterologous origin. In claim 14, a cell wherein the gene encoding the enzyme of i) is derived from rabbit (Oryctolagus cuniculus), chicken (Gallus gallus), cow (Bos Taurus), or pig (Sus scrofa). A cell in claim 7, further comprising a gene encoding the enzyme of iii) and a gene encoding cytochrome p450 oxidoreductase (POR) and cytochrome b5 (CYB5) of homologous or heterologous origin. In claim 7, a cell further comprising a gene encoding the enzyme of iii) from a human (Homo sapiens), rabbit (Oryctolagus cuniculus) or chicken (Gallus gallus) origin, and a gene encoding POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5). In claim 7, a cell further comprising a gene encoding the enzyme of iii) derived from rabbit (Oryctolagus cuniculus) and a gene encoding heterologous POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5). In claim 18, the cell is derived from a brown rat (Rattus norvegicus), a cow (Bos Taurus), or a chicken (Gallus gallus), wherein the genes encoding the heterologous POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5) are derived from a brown rat (Rattus norvegicus), a cow (Bos Taurus), or a chicken (Gallus gallus). In claim 16, a cell in which the gene encoding POR (Cytochrome P450 oxidoreductase) and CYB5 (Cytochrome b5) is expressed simultaneously with the gene encoding the enzyme of i) and / or the gene encoding the enzyme of iii). A cell according to claim 11, further comprising a gene encoding adrenodoxin-NADP+ reductase (FDXR). In claim 21, the cell wherein the gene encoding the adrenodoxin-NADP+ reductase is derived from human (Homo sapiens). In claim 21, a cell wherein the gene encoding the adrenodoxin-NADP+ reductase is expressed simultaneously with the gene encoding the enzyme of viii). In claim 5, a cell wherein the gene encoding the enzyme of i) comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:

2. In claim 6, a cell wherein the gene encoding the enzyme of ii) comprises a nucleotide sequence of SEQ ID NO: 7, 9, 11, 13, 14, 15, 18 or 19. In claim 7, a cell wherein the gene encoding the enzyme of iii) comprises a nucleotide sequence of SEQ ID NO: 20, 22 or 24. In claim 8, a cell wherein the gene encoding the enzyme of iv) comprises a nucleotide sequence of SEQ ID NO: 26, 27 or 31. In claim 9, a cell wherein the gene encoding the enzyme of v) comprises a nucleotide sequence of SEQ ID NO: 35 or 39. In claim 10, a cell wherein the gene encoding the enzyme of vi) comprises a nucleotide sequence of SEQ ID NO: 26, 27 or 31. In claim 11, a cell wherein the gene encoding the enzyme of vii) comprises a nucleotide sequence of SEQ ID NO:

34. In claim 11, a cell wherein the gene encoding the enzyme of viii) comprises a nucleotide sequence of SEQ ID NO:

40. In claim 12, the cell wherein the UPC2-1 gene comprises a nucleotide sequence of SEQ ID NO:

55. In claim 14, the gene encoding the enzyme of i) and the gene encoding cytochrome P450 oxidoreductase (POR) of homologous or heterologous origin comprises the nucleotide sequence of SEQ ID NO: 56, and the gene encoding cytochrome b5 (CYB5) of homologous or heterologous origin comprises the nucleotide sequence of SEQ ID NO:

61. Cell. In claim 16, a cell wherein the gene encoding the enzyme of iii) and the gene encoding cytochrome P450 oxidoreductase (POR) of homologous or heterologous origin comprises the nucleotide sequence of SEQ ID NO: 66, and the gene encoding CYB5 (Cytochrome b5) of homologous or heterologous origin comprises the nucleotide sequence of SEQ ID NO:

71. In claim 21, a cell wherein the gene encoding the FDXR (Adrenodoxin-NADP+ reductase) comprises a nucleotide sequence of SEQ ID NO:

76. A cell according to claim 1, wherein the cell is a yeast cell. A cell according to claim 36, wherein the yeast is Saccharomyces cerevisiae. A method for producing a UDCA precursor using a cell according to any one of claims 1 to 37. In claim 38, the precursor is 7α-hydroxycholesterol, 7α-hydroxycholest-4-en-3-one, 4-cholesten-7α,12α-diol-3-one, 7α,12α-dihydroxy-5β-cholestane-3-one, 3α,7α,12α-trihydroxy-5β-cholestane, 3α,7α,12α,26-tetrahydroxy-5β-cholestane, 3α,7α,12α-trihydroxy-5β-cholestane-26-al, 3α,7α,12α-trihydroxy-5β-cholestanoate, (25R)-3α,7α,12α-trihydroxy-5β-cholestane-26-oil-CoA, (25S)-3α,7α,12α-trihydroxy-5β-cholestane-26-oleoyl-CoA, 3α,7α,12α-trihydroxy-5β-cholestane-24-enoyl-CoA, 3α,7α,12α,24-tetrahydroxy-5β-cholestanoyl-CoA, 3α,7α,12α,trihydroxy-5β-24-oxocholestanoyl-CoA, choleyl-CoA, 7α-hydroxy-5β-cholestane-3-one, 3α,7α-dihydroxy-5β-cholestane, 3α,7α,26-trihydroxy-5β-cholestane, 3α,7α-dihydroxy-5β-cholestane-26-al, A method for producing 3α,7α-dihydroxy-5β-cholestanate, (25R)-3α,7α-dihydroxy-5β-cholestanoyl-CoA, (25S)-3α,7α-dihydroxy-5β-cholestanoyl-CoA, (24E)- 3α,7α-dihydroxy-5β-cholest-24-enoyl-CoA, 3α,7α,24-trihydroxy-5β-cholestanoyl-CoA, 3α,7α-dihydroxy-5β-24-oxocholestanoyl-CoA, chenodeoxycholoyl-CoA, cholic acid (CA) or chenodeoxycholic acid (CDCA).

Images