C-glycosyltransferase mutants and uses thereof

Abstract

The present invention relates to a novel C-glycosyltransferase mutant and its use. The C-glycosyltransferase mutant of the present invention has improved glycosidic bond formation ability compared to wild-type C-glycosyltransferase and can enhance the efficiency of glycoside production of polyketides and similar natural products, particularly types I, II, and III polyketides, non-ribosomal peptides, phenylpropanoids, and other aromatic natural products, and will be useful in the production of drugs, food additives, nutritional supplements, etc. that contain C-glycoside compounds as components.

Description

[Technical Field]

[0001] The present invention relates to novel C-glycosyltransferase mutants and their uses. More specifically, the present invention relates to C-glycosyltransferase mutants characterized by having an amino acid mutation located in the active site of C-glycosyltransferase, thereby enhancing the glycosylation reaction of substrate carbons, and the use of the mutants in the production of polyketide glycosides and phenylpropanoid glycosides. [Background technology]

[0002] Polyketides are a fascinating class of natural products with diverse biological effects and are widely used in food, cosmetics, medicines, and other everyday applications. The enzymes that synthesize polyketides are collectively called polyketide biosynthetic enzymes (PKSs), which are divided into three types: type I, II, and III, depending on the biosynthetic mechanism. Of these, type I PKSs produce macrolide polyketides, while types II and III mainly produce aromatic polyketides.

[0003] For substances with medicinal properties, such as drugs or nutritional supplements, glycosides are generally preferred due to their significantly improved stability, resistance to hydrolysis, and bioavailability compared to non-glycosides. In particular, stable C-glycosidic bonds are chemically more stable than O-glycosidic bonds. However, while several cases of O-glycosylation of natural products in Escherichia coli have been reported (Chen, D.; Chen, R.; Xie, K.; Duan, Y.; Dai, J., Production of acetophenone C-glucosides using an engineered C-glycosyltransferase in Escherichia coli. Tetrahedron Lett. 2018, 59(19), 1875-1878), C-glycosylation has rarely been reported. There have been many reports of O-glycosylation compared to C-glycosylation, not only in E. coli but also in nature.

[0004] Representative C-glycoside natural products include carminic acid and aloesin.

[0005] Carminic acid is a widely used red pigment in foods, cosmetics, and pharmaceuticals. It is extracted directly from scale insects such as cochineal (Dactylopius coccus) and added to foods like ketchup, strawberry milk, and candy, as well as cosmetics like eye shadow, nail polish, and lipstick. However, cochineal grows slowly and only in limited areas (it can only grow in hot, dry regions), limiting its commercial production. The extraction process is also highly inefficient; for example, 70,000 female cochineals are required to produce one pound of carminic acid. Given this situation, the development of a more sustainable method for producing carminic acid was necessary.

[0006] Aloesin is extracted from Aloe vera and is widely used in the cosmetics industry as a skin-whitening agent due to its anti-tyrosinase and anti-melanin production effects. Furthermore, aloesin exhibits anti-inflammatory and anti-radical effects, making it suitable for use as a key ingredient in various medicines and cosmetics. However, the amount of aloesin extracted from aloe plants is extremely small, meaning the development of a more efficient and sustainable bio-based production method was necessary.

[0007] As mentioned above, there is a high demand for C-glycoside natural products, but their supply is insufficient. However, there has been little development of methods for their effective production. In particular, even if attempts were made to produce these compounds through biological processes, efficient production from microbial cell factories was impossible due to the lack of clear enzymes or the low enzymatic conversion efficiency.

[0008] Under these technical backgrounds, the present inventors have made intensive efforts to develop a C-glycosyltransferase with excellent C-glycosylation ability. As a result, they have developed a C-glycosyltransferase mutant that exhibits excellent C-glycosylation ability through amino acid substitution, and confirmed that the C-glycosyltransferase mutant exhibits significantly excellent glycoside production ability for type I, type II, and type III polyketides, nonribosomal peptides, phenylpropanoids, and aromatic natural products in a recombinant microorganism into which the C-glycosyltransferase mutant gene has been introduced, thereby completing the present invention.

[0009] The information provided in this Background section is intended solely to enhance understanding of the background of the present invention and may therefore not include information that constitutes prior art known to those of ordinary skill in the art to which the present invention pertains. [Prior art documents] [Non-patent literature]

[0010] (Non-Patent Document 1) Chen, D. Chen, R.;

[0011] An object of the present invention is to provide novel C-glycosyltransferase mutants and uses thereof.

[0012] In order to achieve the above purpose, The present invention provides a C-glycosyltransferase mutant comprising a mutation in one or more amino acids selected from the group consisting of F17, V93, V132, Y193, L164, and R322 in the C-glycosyltransferase represented by SEQ ID NO: 1.

[0013] The present invention also provides nucleic acids encoding the C-glycosyltransferase mutants.

[0014] The present invention also provides a recombinant microorganism into which the nucleic acid has been introduced.

[0015] The present invention also provides a method for producing a polyketide glycoside and / or a phenylpropanoid glycoside, comprising the steps of: (a) culturing the recombinant microorganism of the present invention to produce polyketide glycosides and / or phenylpropanoid glycosides; (b) recovering the produced polyketide and / or phenylpropanoid products.

[0016] The present invention also provides a method for producing a polyketide glycoside and / or a phenylpropanoid glycoside, comprising the steps of: (a) reacting a C-glycosyltransferase mutant of the present invention or a microorganism expressing the C-glycosyltransferase mutant with a polyketide and / or a phenylpropanoid to produce a polyketide glycoside and / or a phenylpropanoid glycoside; (b) recovering the produced polyketide and / or phenylpropanoid products. [Brief explanation of the drawings]

[0017] FIG. 1 shows the pathway for the production of carminic acid.

[0018] Figure 2 shows the production of flavokermesic acid when different metabolic engineering strategies were introduced. Type II polyketide biosynthetic enzyme (AntDEFBG from P. luminescens) and ZhuIJ produced higher concentrations of FK than type III polyketide biosynthetic enzyme (AaPKS5 from Aloe arborescens) and ZhuIJ.

[0019] FIG. 3 shows the change in the amount of kermesic acid produced by introducing DnrF.

[0020] FIG. 4 shows the candidate C-glycosyltransferases for dcII production and the original enzymatic reaction of each candidate enzyme.

[0021] Figure 5 shows a comparison of the dcII-producing abilities of nine enzyme candidates.

[0022] Figure 6 shows the results of homology modeling and docking simulations to increase KA and dcII production. (a) KA production ability of mutants selected through simulations for DnrF. (b) Protein structure simulation results for the most effective DnrF mutant (P217K). (C) dcII production ability of mutants selected through simulations for GtCGT. (d) Protein structure of the most effective GtCGT mutant (V93Q / Y193F).

[0023] Figure 7 shows the production of carminic acid from glucose. (a) Carminic acid production under different conditions. (b) Analysis of carminic acid through LC-MS / MS analysis. The upper data is the analysis result of commercially available carminic acid, and the lower data is the analysis result of a carminic acid-containing sample produced by E. coli from glucose. The graph on the left is the extracted ion chromatogram (EIC), and the graph on the right is the MS / MS fragmentation pattern. (C) Fed-batch fermentation graph for the final strain. The red arrow indicates the start time of IPTG-mediated gene expression, and DCW indicates dry cell weight.

[0024] FIG. 8 shows the pathway for the production of aloesin.

[0025] Figure 9 shows the production of aloesin via E. coli. (a) Construction and testing of additional plasmids containing RpALS for increased aloesone production. (b) Testing of GtCGT and its mutants for aloesin production. (C) Analysis of aloesin via LC-MS / MS analysis. The top data are from commercial aloesin, and the bottom data are from a sample containing aloesin produced in E. coli from glucose. The graph on the left shows the extracted ion chromatogram, and the graph on the right shows the MS / MS fragment pattern.

[0026] 10 shows the results of testing additional GtCGT mutants to increase aloesin production. The additional mutants were predicted by analyzing the structural model of the GtCGT mutant (V93Q / Y193F).

[0027] 11 shows the results of testing additional GtCGT mutants for increasing aloesin production. The additional mutants were predicted by performing docking simulations based on the GtCGT mutant (V93Q / Y193F). FIG. 12 shows the production (expressed as % conversion) of several phenylpropanoid C-glycosides by the GtCGT mutant (V93Q / Y193F).

[0028] Figure 13 shows the K M and V max A Lineweaver-Burk plot for calculating the values is shown. DISCLOSURE OF THE INVENTION

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. Generally, the nomenclature used herein is that which is well known and commonly used in the art.

[0030] In the present invention, in order to discover C-glycosyltransferase mutants with significantly improved glycosidic bond formation ability compared to the wild-type enzyme, we predicted the protein structure and derived a group of candidate mutants with increased activity through protein structure analysis and computer simulation. Among these, we were able to select effective mutants that have particularly improved substrate binding ability and can enhance the glycosylation reaction.

[0031] Thus, one aspect of the present invention relates to C-glycosyltransferase mutants with improved C-glycosylation ability.

[0032] In the present invention, the C-glycosyltransferase that serves as a template (or wild-type) for the mutant of the present invention refers to an enzyme that induces C-glycosylation by forming a C-glycosidic bond at a carbon atom of a substrate (e.g., a compound, a protein, etc.).

[0033] In the present invention, the C-glycosyltransferase is represented by SEQ ID NO: 1, but is not limited thereto and should be construed as including proteins in which amino acid residues are conservatively substituted at specific amino acid residue positions.

[0034] As used herein, "conservative substitution" refers to a modification of a C-glycosyltransferase that involves replacing one or more amino acids with amino acids having similar biochemical properties that do not result in loss of biological or biochemical function of the C-glycosyltransferase or its variant.

[0035] The term "conservative amino acid substitution" as used herein refers to a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains have been defined and are well known in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0036] Therefore, the C-glycosyltransferase that can be used as a template for the mutants of the present invention is understood to include not only SEQ ID NO: 1, but also all C-glycosyltransferases, recombinant C-glycosyltransferases, and fragments thereof that have substantially the same function and / or effect and have an amino acid sequence identity of 40% or more, 50% or more, 60% or more, 70% or more, preferably 80% or more or 85% or more, more preferably 90% or more, 95% or more, and most preferably 99% or more.

[0037] The term "fragment" of the present invention refers to a fragment obtained by truncating a portion of a parent protein, and may be truncated at the C'-terminus and / or N'-terminus. In the present invention, the fragment refers to a fragment having substantially the same function and / or effect as the deglycosylated C-glycosyltransferase of the present invention. For example, the fragment may include a fragment obtained by truncating the signal sequence of the full-length protein.

[0038] In the present invention, the C-glycosyltransferase may be derived from other strains or other organisms, in addition to GtUF6CGT derived from Gentiana triflora as set forth in SEQ ID NO: 1. For example, IroB (EnCGT) derived from E. coli Nissle. The enzyme may be, but is not limited to, UGT708A6 (ZmCGT) dual C / O-glycosyltransferase derived from Zea mays; UGT708C2 (FeCGT) derived from Fagopyrum esculentum; MiCGT derived from Mangifera indica; OsCGT derived from Oryza sativa; UGT708D1 (GmCGT) derived from Glycine max; GtUF6CGT1 (GtCGT) derived from Gentiana triflora; or AvCGT derived from Aloe vera, and preferably GtUF6CGT1 (GtCGT) derived from Gentiana triflora or UGT708A6 (ZmCGT) dual C / O-glycosyltransferase derived from Zea mays.

[0039] In one embodiment of the present invention, it was confirmed that when a mutant was generated by substituting some of the amino acids of a wild-type C-glycosyltransferase, it exhibited significantly superior C-glycosylation induction ability, and when the C-glycosyltransferase was introduced into a recombinant strain for polyketide synthesis, C-glycosylated polyketides could be produced in significant yields.

[0040] In the present invention, the C-glycosyltransferase mutant can be characterized by containing a mutation in one or more amino acids selected from the group consisting of F17, V93, V132, Y193, L164 and R322 in the C-glycosyltransferase represented by SEQ ID NO: 1, and more preferably, by containing a mutation in the amino acid V93 and / or Y193.

[0041] In the present invention, the C-glycosyltransferase mutant can be characterized in that, in addition to any one or more amino acids selected from the group consisting of F17, V93, V132, Y193, L164, and R322, it contains a mutation in one or more other amino acids in the C-glycosyltransferase represented by SEQ ID NO: 1.

[0042] In the present invention, the C-glycosyltransferase mutant is a C-glycosyltransferase represented by SEQ ID NO: 1 that has the following amino acids: F17, V405, P107, L208, L164, P45, I305, L316, F401, Y94, N57, Y187, C16, P319, F167, V132, N206, R406, Q386, V129, L125, L194, I9 5, S215, L184, Y158, L29, L27, F202, H159, S370, H365, V329, M301, V315, V190, C366, W80, L58, Q210, F 312, D61, I207, L363, P196, L106, V93, A394, W314, S155, P88, D99, Y284, E189, G49, H328, E399, T392, F 387, A44, P199, E46, R28, V285, I124, R419, L306, Y157, Y200, E373, P191, L214, S376, V15, E332, E51, I417, L98, I323, H161, T383, P127, E309, N84, L313, Q104, T371, N213, G79, L330, N307, K105, L128, A15 The amino acid sequence may further include a mutation at any one or more amino acids selected from the group consisting of: 2, I18, N59, W147, S86, L293, E296, S377, L185, K216, F89, S286, F396, F211, Y303, D223, R415, N96, V22, S153, F154, D192, Y193, H195, P201, Y292, and R322.

[0043] In the present invention, the C-glycosyltransferase mutant may further include a mutation in any one or more amino acids selected from the group consisting of I18, Q20, T50, I95, V290, I323, V22, L29, E46, V48, E51, A55, S86, D99, R103, C151, L184, L194, E332, and P385 in the C-glycosyltransferase represented by SEQ ID NO: 1.

[0044] In the present invention, preferably, the C-glycosyltransferase mutant can further include a mutation in any one or more amino acids selected from the group consisting of I323, T50, I18, I95, Q20, P385, L194, and V48 in the C-glycosyltransferase represented by SEQ ID NO: 1.

[0045] The term "mutant" of the present invention is used to refer to a mutation of some amino acid residues in the amino acid sequence of a reference sequence (e.g., a normal C-glycosyltransferase sequence, SEQ ID NO: 1), preferably substitution, deletion, and / or insertion of amino acid residues, more preferably substitution of amino acid residues, as well as deletion of some amino acid residues at the N-terminus or C-terminus in addition to such substitution, deletion, and / or insertion of amino acid residues. In one embodiment of the present invention, the mutant was prepared by substituting some amino acids in SEQ ID NO: 1, but is not limited thereto.

[0046] In the present invention, the "mutation" can be characterized as an amino acid substitution.

[0047] In the present invention, the C-glycosyltransferase mutant can be characterized in that it contains one or more amino acid substitutions selected from the group consisting of F17G, V93Q, V132A, Y193F, L164G and R322D in the C-glycosyltransferase represented by SEQ ID NO: 1, more preferably V93Q and / or Y193F, and most preferably V93Q and Y193F.

[0048] In the present invention, the C-glycosyltransferase mutant can be characterized in that in addition to one or more amino acid substitutions selected from the group consisting of F17G, V93Q, V132A, Y193F, L164G, and R322D, it further contains one or more other amino acid substitutions in the C-glycosyltransferase represented by SEQ ID NO: 1.

[0049] In the present invention, the C-glycosyltransferase mutant can be characterized in that in addition to the V93Q and Y193F amino acid substitutions in the C-glycosyltransferase represented by SEQ ID NO: 1, it further contains one or more other amino acid substitutions.

[0050] In the present invention, the C-glycosyltransferase mutant may further include other amino acid substitutions such as F17G, V405M, P107G, L208G, L164G, P45G, I305A, L316G, F401H, Y94G, N57G, Y187A, C16G, P319G, F167G, V132A, N206E, R406G, Q386H, V129A, L125V, L194A, and I95G in the C-glycosyltransferase represented by SEQ ID NO: 1. , S215D, L184G, Y158T, L29A, L27A, F202S, H159G, S370A, H365G, V329T, M301W, V315A, V190A, C366G, W80Y, L58E, Q210G, F312G, D6 1G, I207P, L363G, P196G, L106G, V93G, A394G, W314C, S155A, P88D, D99G, Y284H, E189A, G49TH328G, E399D, T392A, F387T, A44G, P19 9E, E46G, R28G, V285I, I124T, R419A, L306M, Y157T, Y200L, E373A, P201G, P191G, L214A, S376G, V15G, E332P, E51C, I417L, L98G, I 323A, H161G, T383C, P127A, E309N, N84S, L313T, Q104D, T371A, N213L, G79S, L330G, N307A, K105G, L128D, A152G, S153G, I18A, N59V , W147F, S86V, L293V, E296D, S377A, L185V, K216R, F89A, S286C, F396L, F211G, Y303A, D223G, R415L, N96A, V22H, V93Q, V93L, S153C, F154L, D192S, Y193F, H195Y, H195L, P201T, Y292H, Y292F, R322D and R322A.

[0051] In the present invention, the additional amino acid substitutions that may be included may be characterized as being any one or more amino acid substitutions selected from the group consisting of I18P, Q20M, T50N, T50Q, T50K, T50R, T50V, I95M, I95T, V290G, V290A, I323S, I323A, I95L, V22A, L29A, E46G, V48G, E51C, A55S, S86V, D99G, R103V, C151G, L184G, L194A, E332P, I18A, and P385A in the C-glycosyltransferase represented by SEQ ID NO: 1.

[0052] In the present invention, the additional amino acid substitution that may be included may preferably be characterized as being one or more amino acid substitutions selected from the group consisting of I323S, T50R, T50V, I18P, I95T, Q20M, I323A, P385A, L194A, and V48G in the C-glycosyltransferase represented by SEQ ID NO: 1.

[0053] In one embodiment of the present invention, i) V93Q and Y193F amino acid substitutions in the C-glycosyltransferase represented by SEQ ID NO: 1; ii) V93Q, Y193F and I323S amino acid substitutions in the C-glycosyltransferase represented by SEQ ID NO: 1; or iii) It was confirmed that, in the C-glycosyltransferase represented by SEQ ID NO: 1, C-glycosyltransferase mutants containing the amino acid substitutions V93Q, Y193F, and P385A exhibited the most efficient C-glycosylation, but are not limited thereto.

[0054] The positions of the amino acid residues of the amino acid mutations can be precisely numbered using the amino acid sequence set forth in SEQ ID NO: 1 as a reference, where "residue Xn" refers to residue X corresponding to position n in the amino acid sequence set forth in SEQ ID NO: 1, where n is a positive integer and X is an abbreviation for any amino acid residue. For example, "residue V93" refers to amino acid residue V corresponding to position 93 in the amino acid sequence set forth in SEQ ID NO: 1.

[0055] In the present invention, an "amino acid mutation" may be an "amino acid substitution Xn," which in one embodiment refers to an amino acid substitution occurring at amino acid residue X at position n in the amino acid sequence represented by SEQ ID NO: 1, where n is a positive integer and X is an abbreviation for any amino acid residue. For example, "amino acid substitution V93" refers to an amino acid substitution occurring at amino acid residue V corresponding to position 93 in the amino acid sequence represented by SEQ ID NO: 1.

[0056] In the present invention, when a C-glycosyltransferase having an amino acid sequence other than that of SEQ ID NO: 1 is used as a reference sequence, the amino acid residue "corresponding" to a specific amino acid residue described with reference to SEQ ID NO: 1 is generally obtained by aligning the amino acid sequences under optimized conditions. The sequence alignment can be performed by means understood by those skilled in the art, such as using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters to use for alignment, including any algorithms necessary to achieve optimal alignment of the full-length sequences being compared.

[0057] The amino acid substitutions of the present invention may be non-conserved substitutions, which may involve altering an amino acid residue in a target protein or polypeptide in a non-conservative manner, such as replacing an amino acid residue having a particular side chain size or particular properties (e.g., hydrophilicity) with an amino acid residue having a different side chain size or different properties (e.g., hydrophobicity).

[0058] The amino acid substitutions may also be conservative substitutions. Conservative substitutions may involve changing the amino acid residues of a target protein or polypeptide in a conservative manner, such as replacing an amino acid residue with a specific side chain size or specific characteristics (e.g., hydrophilicity) with an amino acid residue with the same or similar side chain size or the same or similar properties (e.g., still hydrophilicity). Such conservative substitutions generally do not significantly affect the structure or function of the resulting protein. In the present application, amino acid sequence variants that are mutations of fusion proteins, fragments thereof, or variants in which one or more amino acids are substituted may include conservative amino acid substitutions that do not significantly change the structure or function of the protein.

[0059] For example, mutual substitutions between amino acids in each of the following groups can be considered conservative substitutions in this application: Amino acid groups with non-polar side chains: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan and methionine. Uncharged amino acid groups with polar side chains: glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Negatively charged amino acid groups with polar side chains: aspartic acid and glutamic acid. Positively charged basic amino acid groups: lysine, arginine and histidine. Amino acid groups containing phenyl: phenylalanine, tryptophan and tyrosine.

[0060] The proteins, polypeptides and / or amino acid sequences encompassed by the present invention may also be understood to encompass at least the following: variants or homologues having the same or similar function as said proteins or polypeptides.

[0061] In the present invention, the variant can be a protein or polypeptide generated by the substitution, deletion, or addition of one or more amino acids compared to the amino acid sequence of a wild-type C-glycosyltransferase. For example, the functional variant can include a protein or polypeptide having an amino acid change due to the substitution, deletion, and / or insertion of at least one amino acid, e.g., 1 to 30, 1 to 20, or 1 to 10, alternatively, e.g., 1, 2, 3, 4, or 5 amino acids. The functional variant can substantially retain the biological properties of the protein or polypeptide prior to the alteration (e.g., substitution, deletion, or addition). For example, the functional variant can retain 60%, 70%, 80%, 90%, or 100% or more of the biological activity of the protein or polypeptide prior to the alteration.

[0062] In the present invention, the homologue may be a protein or polypeptide having at least about 80% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or polypeptide.

[0063] In the present invention, the homology generally refers to the similarity, significance, or association between two or more sequences. "Percentage of sequence homology" can be calculated by comparing two aligned sequences within a comparison window to determine the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) is present, and the number of matching positions within the comparison window (i.e., window size) is determined by dividing the number of matching positions by the total number of positions, and multiplying the result by 100 to provide the percentage of sequence homology. Alignment to determine the percentage of sequence homology can be performed in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithm required to achieve maximum alignment within the full-length sequence or target sequence region being compared.The homology can also be determined by the following methods: FASTA and BLAST.The FASTA algorithm is disclosed, for example, in W.R. Pearson and D.J. Lipman's "Improved Tool for Biological Sequence Comparison," Proc. Natl. Acad. Sci., 85:2444-2448, 1988; and D.J. Lipman and W.R. Pearson's "Fast and Sensitive Protein Similarity Search," Science, 227:1435-1441, 1989; and for a description of the BLAST algorithm, see S. Altschul, W. Gish, W. Miller, E.W. Myers and D. Lipman, "A Basic Local Alignment Search Tool," Journal of Molecular Biology, 215:403-410, 1990.

[0064] In the present invention, the C-glycosyltransferase mutant can be characterized by having enhanced glycosylation of substrate carbon compared to the wild-type.

[0065] In the present invention, the mutated amino acid is located in the active site of the enzyme, and the substrate binding ability of the mutant is improved by 10% or more, preferably 20% or more, and more preferably 50% or more compared to the wild type through the mutation of the amino acid.

[0066] In one embodiment of the present invention, it has been confirmed that when the C-glycosyltransferase mutant of the present invention is used with various polyketide compounds (flavokermesic acid, kermesic acid, aloesone) or phenylpropanoid compounds (naringenin, apigenin, or luteolin) as substrates, significantly higher C-glycosylation rates can be achieved compared to the wild-type enzyme, regardless of the type of substrate. Therefore, the C-glycosyltransferase mutant of the present invention can be used to C-glycosylate various compounds or proteins as substrates. For example, the C-glycosyltransferase mutant of the present invention can be characterized as being used for the C-glycosylation of the polyketide compounds or phenylpropanoid compounds, but is not limited thereto.

[0067] In the present invention, the substrate may be any polyketide or phenylpropanoid compound without limitation, and may preferably be, as confirmed in one embodiment, flavokermetic acid, kermesic acid, aloesone, naringenin, apigenin, or luteolin, but is not limited thereto.

[0068] The substrate is preferably flavokermetic acid or kermesic acid, and the mutant can be characterized by glycosylation of the second carbon of flavokermetic acid, but is not limited thereto.

[0069] The substrate is preferably aloezone, and the mutant can be characterized by, but is not limited to, glycosylation of the eighth carbon of the aloezone.

[0070] In another aspect, the present invention relates to a nucleic acid encoding the C-glycosyltransferase mutant.

[0071] From another aspect, the present invention relates to a vector containing the nucleic acid.

[0072] From another aspect, the present invention relates to a recombinant microorganism into which the nucleic acid has been introduced.

[0073] In the present invention, the recombinant microorganism may be characterized in that the nucleic acid is introduced into the host microorganism in the form of a plasmid or inserted into the genome.

[0074] In the present invention, the recombinant microorganism may be characterized as being for producing polyketide glycosides and / or phenylpropanoid glycosides, but is not limited thereto.

[0075] In the present invention, the recombinant microorganism can be characterized by having the ability to produce polyketides and / or phenylpropanoids as substrates for the C-glycosyltransferase of the present invention, and the polyketides and / or phenylpropanoids can be glycosylated by the C-glycosyltransferase expressed by the recombinant microorganism of the present invention and converted into polyketide glycosides and / or phenylpropanoid glycosides.

[0076] In the present invention, the polyketide is a type I polyketide selected from the group consisting of rapamycin, lovastatin, erythromycin, rifamycin, avermectin, geldanamycin, ivermectin, calicheamicin, epothilone, triacetic acid lactone, and 6-methylsalicylic acid; a type II polyketide selected from the group consisting of actinorhodin, doxorubicin, daunorubicin, oxytetracycline, SEK4, SEK4b, SEK34, SEK15, SEK26, FK506, DMAC, aklavinone, aklanonic acid, epsilon-rhodomycinone, doxycycline, anthramycin, tetracenomycin, carminic acid, and frenolicin; and type III polyketides selected from the group consisting of aloesin, aloenin, barbaloin, 5,7-dihydroxy-2-methylchromone, and aloesone, but are not limited thereto.

[0077] In the present invention, the phenylpropanoid is Actinomycin, bacitracin, daptomycin, vancomycin, teixobactin, tyrocidine, gramicidin, zwittermicin A, bleomycin, cyclosporin, pyoverdine, enterobactin, myxochelin A A), indigoidine, non-ribosomal peptides such as cyanophycin, pinocembrin, dihydrokaempferol, eriodictyol, dihydroquercetin, coniferyl alcohol alcohol), silibinin, isosilybin, silychristin, silinide, 2,3-dihydrosilybin, silydianin, daidzein, genistein, apigenin, luteolin, kaempferol, quercetin, catechin, pelargonidin The catechin may be selected from the group consisting of, but not limited to, cyanidin, afzelechin, myricetin, fisetin, galangin, hesperetin, tangeritin, delphinidin, epicatechin, chrysin, resveratrol, and naringenin.

[0078] In the present invention, the host microorganism can be characterized as having the ability to produce a precursor of the polyketide glycoside and / or phenylpropanoid glycoside to be produced.

[0079] In the present invention, the precursor of the polyketide glycoside and / or phenylpropanoid glycoside may be a polyketide and / or phenylpropanoid, preferably a non-glycosylated polyketide and / or phenylpropanoid.

[0080] In the present invention, the host microorganism may be characterized as a microorganism that naturally produces precursors of the polyketide glycosides and / or phenylpropanoid glycosides, or a recombinant microorganism that has been produced by genetic engineering to produce precursors of the polyketide glycosides and / or phenylpropanoid glycosides.

[0081] In the present invention, the recombinant microorganism may be characterized by enhanced production of nucleotides, preferably NTP-sugars, to improve the conversion rate of glycosides by the introduced C-glycosyltransferase. For example, the recombinant microorganism may be further characterized by enhanced expression of genes encoding UTP-glucose-1-phosphate uridylyltransferase, phosphoglucomutase, and / or nucleoside-diphosphate kinase, but is not limited thereto.

[0082] In the present invention, the UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase, and / or nucleoside diphosphate kinase may be derived from, but is not limited to, E. coli, and the expression of genes involved in the production of NTP-sugars may be enhanced depending on the host strain.

[0083] In one embodiment of the present invention, flavokermetic acid, kermetic acid, aloesone, naringenin, apigenin or luteolin was used as the precursor of the polyketide glycoside and / or phenylpropanoid glycoside, but the precursor is not limited thereto, and the precursors of the various polyketide glycosides and / or phenylpropanoid glycosides described above are well known in the art and can be easily selected from them.

[0084] The term "nucleic acid" of the present invention generally refers to an isolated form of a nucleotide, deoxyribonucleotide, or ribonucleotide, or analogue thereof of any length, isolated from its natural environment or artificially synthesized. The nucleic acids of the present invention can be isolated. For example, they can be produced or synthesized by the following methods: (i) in vitro amplification, such as polymerase chain reaction (PCR) amplification; (ii) clonal recombination; (iii) purification, e.g., fractionation by restriction enzyme digestion and gel electrophoresis; or (iv) synthesis, e.g., chemical synthesis. In some embodiments, the isolated nucleic acid is a nucleic acid molecule produced by recombinant DNA technology. In the present invention, nucleic acids encoding the variants can be produced by various methods known in the art. Such methods include, but are not limited to, restriction fragment manipulation or overlap extension PCR using synthetic oligonucleotides. The manufacturing method and principles can be found in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, NY, 1993.

[0085] The term "plasmid" as used herein may be used interchangeably with "vector" and generally refers to a nucleic acid molecule capable of transferring inserted nucleic acid to a host cell (including a host microorganism) and autonomously replicating in the host cell or microorganism. Examples of such vectors include vectors primarily used to insert DNA or RNA into cells, vectors primarily used for DNA or RNA replication, and vectors primarily used for transcriptional and / or translational expression of DNA or RNA. The term also includes vectors with multiple functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into an appropriate host cell. Generally, the vector can produce a desired expression product by culturing an appropriate host cell containing the vector. In the present invention, the vector may contain one or more of the nucleic acids. For example, the vector may contain all the nucleic acid molecules necessary to encode the mutant. In this case, only one vector is required to obtain the fusion protein of the present application. In some embodiments, the vector may contain a nucleic acid molecule encoding a portion of the mutant. Alternatively, the vector may contain, for example, a nucleic acid molecule for regulating gene expression in the recombinant microorganism. In this case, two or more different vectors may be required to obtain the recombinant microorganism of the present invention.

[0086] The vector may also contain other genes, such as marker genes, for selecting the vector in an appropriate host cell under appropriate conditions. Furthermore, the vector may also contain expression control elements that ensure the appropriate expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequence is a regulatory element. The specific structure of the expression control sequence may vary depending on the species or cell type, but generally includes 5' non-transcribed sequences involved in initiation of transcription and translation, such as a TATA box, capping sequences, CAAT sequences, etc., as well as 5' and 3' non-translated sequences. For example, the 5' non-transcribed expression control sequence may include a promoter region, which may include a promoter sequence for regulating transcription of an operably linked nucleic acid. In the present invention, the vector can be selected from the group consisting of pET-30a-c(+), pET-22b(+), pCDFDuet-1, pACYCDuet-1, pRSFDuet-1, pBBR1MCS, pSC101, pTac15K, pTrc99A, pCOLADuet-1 and pBR322, but is not limited thereto, and a person skilled in the art can appropriately select and use vectors commonly used in this technical field in addition to the above vectors.

[0087] The terms "host cell," "cell," "host microorganism," and "host" of the present invention can be used interchangeably and generally refer to an individual cell, cell line, microorganism, or cell culture that contains or can contain a nucleic acid of the present invention, or a plasmid or vector, or that can express a mutant or expression-regulated protein or polypeptide of the present invention. The host cell can include the progeny of a single host cell. Due to spontaneous, accidental, or deliberate mutations, the progeny cells and the original mother cell are not necessarily completely identical in morphology or genome, as long as they are capable of expressing the target protein or polypeptide of the present invention. The host cell can be obtained by transfecting in vitro cells with the vector of the present invention.The host cell may preferably be a microorganism, such as E. coli, Rhizobium, Bifidobacterium, Candida, Erwinia, Enterobacter, Pasteurella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Azotobacter, Acinetobacter, Ralstonia, Agrobacterium, etc. The bacterial strain may be selected from the group consisting of, but not limited to, Bacillus, Staphylococcus, Lactococcus, Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium, Cyanobacterium, and Cyclobacterium.

[0088] Meanwhile, in the present invention, it was confirmed that various polyketide glycosides or phenylpropanoid glycosides can be effectively produced using recombinant microorganisms capable of expressing the C-glycosyltransferase mutants.

[0089] Therefore, from yet another aspect, the present invention relates to a recombinant microorganism for producing polyketide glycosides or phenylpropanoid glycosides, into which a nucleic acid encoding the C-glycosyltransferase mutant of the present invention has been introduced.

[0090] In the present invention, the polyketide glycoside may be a type I polyketide glycoside, a type II polyketide glycoside, or a type III polyketide glycoside.

[0091] In the present invention, the polyketide is a type I polyketide selected from the group consisting of rapamycin, lovastatin, erythromycin, rifamycin, avermectin, geldanamycin, ivermectin, calicheamicin, epothilone, triacetic acid lactone, and 6-methylsalicylic acid;

[0092] a type II polyketide selected from the group consisting of actinorhodin, doxorubicin, daunorubicin, oxytetracycline, SEK4, SEK4b, SEK34, SEK15, SEK26, FK506, DMAC, aklavinone, aklanonic acid, epsilon-rhodomycinone, doxycycline, anthramycin, tetracenomycin, carminic acid, and frenolicin;

[0093] and type III polyketides selected from the group consisting of aloesin, aloenin, barbaloin, 5,7-dihydroxy-2-methylchromone, and aloesone, but are not limited thereto.

[0094] In the present invention, the recombinant microorganism for producing a polyketide glycoside or a phenylpropanoid glycoside may be characterized by producing a precursor of each glycoside. For example, the recombinant microorganism may be characterized by producing a polyketide or a phenylpropanoid, which is a precursor of each glycoside.

[0095] In the present invention, the recombinant microorganism for producing a polyketide glycoside or a phenylpropanoid glycoside can be characterized by producing a polyketide or a phenylpropanoid by introducing an additional gene. Recombinant microorganisms capable of synthesizing a polyketide by introducing a gene can be produced, for example, by the genes and methods described in the present inventors' published paper, Yang, D., Kim, WJ, Yoo, SM, Choi, JH, Ha, SH, Lee, MH, and Lee, SY, "Repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria," Proc. Natl. Acad. Sci. (PNAS), 115 (40) 9835-9844 (https: / / doi.org / 10.1073 / pnas.1808567115) (October 2, 2018), and Korean Patent Registration No. 10-2187682, but are not limited thereto. A person skilled in the art can construct recombinant microorganisms capable of synthesizing polyketides or phenylpropanoids by introducing various polyketide or phenylpropanoid synthesis pathways and genes involved therein described in the art into various host microorganisms.

[0096] In the present invention, the recombinant microorganism for producing a polyketide glycoside or a phenylpropanoid glycoside can be further characterized in that a polyketide synthase or a phenylpropanoid synthase has been introduced into the recombinant microorganism.

[0097] In the present invention, the polyketide synthase may be, for example, a type I polyketide synthase, a type II polyketide synthase, or a type III polyketide synthase, but is not limited thereto.

[0098] In the present invention, when the recombinant microorganism for producing the polyketide glycoside or phenylpropanoid glycoside does not produce a precursor of each glycoside, the polyketide glycoside or phenylpropanoid glycoside can be produced by adding a precursor of each glycoside to the culture medium.

[0099] In the present invention, the recombinant microorganism can be characterized as being for producing type I polyketide glycosides.

[0100] In the present invention, the recombinant microorganism for producing a type I polyketide glycoside may be characterized by producing a precursor of the type I polyketide glycoside. For example, the precursor of the type I polyketide glycoside may be, but is not limited to, rapamycin, lovastatin, erythromycin, rifamycin, etc.

[0101] In the present invention, the recombinant microorganism for producing type I polyketide glycosides can be characterized by producing a precursor of type I polyketide glycosides by introducing an additional gene. In the present invention, the recombinant microorganism for producing type I polyketide glycosides is, for example, (i) It can be characterized in that a gene encoding a type I polyketide biosynthetic enzyme is further introduced. In the present invention, the type I polyketide biosynthetic enzymes can be easily selected from various protein and gene databases.

[0102] Therefore, a host microorganism into which a nucleic acid encoding a C-glycosyltransferase mutant of the present invention and a type I polyketide biosynthetic enzyme gene are introduced can be characterized as having the ability to produce coenzyme A, preferably malonyl-CoA or acetyl-CoA.

[0103] Therefore, in the present invention, the recombinant microorganism may be characterized by enhanced production of coenzyme A. For example, in the present invention, the recombinant microorganism may be further characterized by (ii) suppressed or attenuated expression of the pabA gene, but is not limited thereto. Recombinant microorganisms with enhanced coenzyme A production can be produced using various strategies for mass production of coenzyme A known in the art.

[0104] In the present invention, the recombinant microorganism may be characterized by enhanced production of nucleotides, preferably NTP-sugars, to improve the glycoside conversion rate by the introduced C-glycosyltransferase. For example, in the present invention, the recombinant microorganism may be further characterized by, but not limited to, (iii) enhanced expression of genes encoding UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase, and / or nucleoside diphosphate kinase.

[0105] In the present invention, the recombinant microorganism may be characterized as being for producing a type II polyketide glycoside, for example, but not limited to, carminic acid.

[0106] In the present invention, the recombinant microorganism for producing a type II polyketide glycoside may be characterized by producing a precursor of the type II polyketide glycoside, for example, the precursor of the type II polyketide glycoside may be, but is not limited to, flavochermic acid or chermic acid.

[0107] In the present invention, the recombinant microorganism for producing a type II polyketide glycoside can be characterized in that it produces a precursor of a type II polyketide glycoside by introducing an additional gene.

[0108] In the present invention, the recombinant microorganism for producing type II polyketide glycosides can be characterized in that (i) a gene encoding a type II polyketide biosynthetic enzyme is further introduced.

[0109] In the present invention, the recombinant microorganism for producing the type II polyketide glycoside, preferably carminic acid, is, for example, (i) a gene encoding a type II polyketide biosynthetic enzyme; (ii) a gene encoding 4'-phosphopantetheinyl transferase; and (iii) a gene encoding a cyclase; (iv) a gene encoding acetyl-CoA carboxylase; (v) The plant may be characterized in that any one or more genes selected from the group consisting of genes encoding aklavinone 12-hydroxylase are further introduced, and preferably, all of the genes are introduced.

[0110] As shown in Figure 1, type II polyketides, which are substrates of the C-glycosyltransferase of the present invention, can be converted from coenzyme A (CoA) such as malonyl-CoA or acetyl-CoA by the enzyme encoded by the introduced gene. Therefore, a host microorganism into which the nucleic acid encoding the C-glycosyltransferase mutant and the type II polyketide biosynthetic enzyme gene or the genes (i) to (v) are introduced can be characterized as having the ability to produce coenzyme A, preferably malonyl-CoA or acetyl-CoA.

[0111] In an embodiment of the present invention, it was confirmed that suppression or attenuation of the expression of the pabA gene leads to the accumulation of coenzyme A, resulting in improved synthesis of polyketides, which are precursors of the C-glycosyltransferase of the present invention.

[0112] Therefore, in the present invention, the recombinant microorganism may be characterized by enhanced production of coenzyme A. For example, in the present invention, the recombinant microorganism may be further characterized by (ii) suppressed or attenuated expression of the pabA gene, but is not limited thereto. Recombinant microorganisms with enhanced coenzyme A production can be produced using various strategies for mass production of coenzyme A known in the art.

[0113] In the present invention, the recombinant microorganism may be characterized by enhanced production of nucleotides, preferably NTP-sugars, to improve the glycoside conversion rate by the introduced C-glycosyltransferase. For example, in the present invention, the recombinant microorganism may be further characterized by, but not limited to, (iii) enhanced expression of genes encoding UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase, and / or nucleoside diphosphate kinase.

[0114] In the present invention, the UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase, and / or nucleoside diphosphate kinase may be derived from, but is not limited to, E. coli, and the expression of genes involved in the production of NTP-sugars may be enhanced depending on the host strain.

[0115] In the present invention, the gene encoding the polyketide biosynthesis enzyme II may be characterized as being any one or more genes selected from the group consisting of antD (ketosynthase), antE (chain-length factor), antF (ACP), antB (phosphopantetheinyl transferase), and antG (malonyl-CoA:ACP malonyltransferase), or a combination thereof, but is not limited thereto.

[0116] In the present invention, the aklavineone 12-hydroxylase may be characterized by, but is not limited to, a mutation of the 217th amino acid in the amino acid sequence shown in SEQ ID NO: 2 from proline to lysine (P217K).

[0117] In the present invention, the type II polyketide biosynthetic enzyme is derived from P. luminescens, The 4'-phosphopantetheinyl transferase is derived from Bacillus subtilis or P. luminescens, The cyclase is derived from Streptomyces sp. the acetyl-CoA carboxylase is from Corynebacterium glutamicum, and / or The acravineone 12-hydroxylase may be characterized as being derived from Streptomyces peucetius, but is not limited thereto.

[0118] In the present invention, the recombinant microorganism may be characterized as being used for producing a type III polyketide glycoside, for example, but not limited to, aloesin.

[0119] In the present invention, the recombinant microorganism for producing a type III polyketide glycoside may be characterized by producing a precursor of the type III polyketide glycoside, for example, but not limited to, aloezone.

[0120] In the present invention, the recombinant microorganism for producing a type III polyketide glycoside can be characterized in that it produces a precursor of a type III polyketide glycoside by introducing an additional gene.

[0121] In the present invention, a recombinant microorganism for producing type III polyketide glycosides is, for example, (i) The plant may be characterized in that a gene encoding a type III polyketide biosynthetic enzyme is introduced into the plant. For example, the type III polyketide biosynthetic enzyme may be, but is not limited to, aloezone synthase.

[0122] In the present invention, the aloezone synthase may be characterized as being derived from R. palmatum, but is not limited thereto.

[0123] As shown in Figure 8, a type III polyketide (e.g., aloezone), which is a substrate of the C-glycosyltransferase of the present invention, can be converted from coenzyme A (CoA) such as malonyl-CoA or acetyl-CoA by the enzyme encoded by the introduced gene to produce the type III polyketide, which is a substrate of the C-glycosyltransferase of the present invention. Therefore, a host microorganism into which a nucleic acid encoding a C-glycosyltransferase mutant and a type III polyketide biosynthetic enzyme gene are introduced can be characterized as having the ability to produce coenzyme A, preferably malonyl-CoA or acetyl-CoA.

[0124] Therefore, in the present invention, the recombinant microorganism may be characterized by enhanced production of coenzyme A. For example, in the present invention, the recombinant microorganism may be further characterized by, but not limited to, (ii) suppressed or attenuated expression of the pabA gene, and recombinant microorganisms with enhanced coenzyme A production may be produced using various strategies for mass production of coenzyme A known in the art.

[0125] In the present invention, the recombinant microorganism may be characterized by enhanced production of nucleotides, preferably NTP-sugars, to improve the glycoside conversion rate by the introduced C-glycosyltransferase. For example, the recombinant microorganism may be further characterized by enhanced expression of genes encoding UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase, and / or nucleoside diphosphate kinase, but is not limited thereto.

[0126] In the present invention, the UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase, and / or nucleoside diphosphate kinase may be derived from, but is not limited to, E. coli, and the expression of genes involved in the production of NTP-sugars may be enhanced depending on the host strain.

[0127] In the present invention, the recombinant microorganism may be used for producing a phenylpropanoid glycoside, for example, but not limited to, vitexin, naringenin-6-C-glucoside, or isoorientin.

[0128] In the present invention, the recombinant microorganism for producing a phenylpropanoid glycoside may be characterized by producing a precursor of the phenylpropanoid glycoside, for example, but not limited to, apigenin, naringenin, or luteolin.

[0129] In the present invention, the recombinant microorganism for producing phenylpropanoid glycosides can be characterized in that it produces precursors of phenylpropanoid glycosides by introducing an additional gene into the microorganism.

[0130] In the present invention, the recombinant microorganism for producing phenylpropanoid glycosides is, for example, (i) It can be characterized in that a gene encoding a phenylpropanoid biosynthetic enzyme is further introduced.

[0131] Phenylepropanoids, which are substrates for the C-glycosyltransferase of the present invention, can be converted from coenzyme A (Coenzyme A-CoA) such as malonyl-CoA or aromatic CoA (e.g., coumaroyl-CoA) by the enzyme encoded by the introduced gene. Therefore, a host microorganism into which the nucleic acid encoding the C-glycosyltransferase mutant and the phenylpropanoid biosynthetic enzyme gene are introduced can be characterized as having the ability to produce coenzyme A, preferably malonyl-CoA or coumaroyl-CoA.

[0132] Therefore, in the present invention, the recombinant microorganism may be characterized by enhanced production of coenzyme A. For example, in the present invention, the recombinant microorganism may be further characterized by (ii) suppressed or attenuated expression of the pabA gene, but is not limited thereto. Recombinant microorganisms with enhanced coenzyme A production can be produced using various strategies for mass production of coenzyme A known in the art.

[0133] In the present invention, the recombinant microorganism may be characterized by enhanced production of nucleotides, preferably NTP-sugars, to improve the glycoside conversion rate by the introduced C-glycosyltransferase. For example, in the present invention, the recombinant microorganism may be further characterized by, but not limited to, (iii) enhanced expression of genes encoding UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase, and / or nucleoside diphosphate kinase.

[0134] In the present invention, the UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase, and / or nucleoside diphosphate kinase may be derived from, but is not limited to, E. coli, and the expression of genes involved in the production of NTP-sugars may be enhanced depending on the host strain.

[0135] For example, the recombinant microorganism of the present invention is a recombinant microorganism into which a nucleic acid encoding a C-glycosyltransferase of the present invention has been introduced, (i) introduction of genes encoding type II polyketide biosynthetic enzymes; (ii) introduction of a gene encoding 4'-phosphopantetheinyl transferase; (iii) introduction of a gene encoding a cyclase; (iv) introduction of a gene encoding acetyl-CoA carboxylase; (v) introduction of a gene encoding aklavineone 12-hydroxylase; (vi) enhanced expression of genes encoding UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase, and / or nucleoside diphosphate kinase; (vii) attenuation of expression of the pabA gene.

[0136] In another example, the recombinant microorganism of the present invention is a recombinant microorganism into which a nucleic acid encoding a C-glycosyltransferase of the present invention has been introduced, (i) introduction of a gene encoding aloezone synthase; (ii) attenuation of the expression of the pabA gene, and (iii) enhanced expression of a gene encoding glucose 6-phosphate 1-dehydrogenase.

[0137] In another example, the recombinant microorganism of the present invention is a recombinant microorganism into which a nucleic acid encoding a C-glycosyltransferase of the present invention has been introduced,

[0138] The recombinant microorganism may be characterized as a recombinant microorganism for producing polyketide glycosides or phenylpropanoid glycosides, in which expression of a gene encoding UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase, and / or nucleoside diphosphate kinase is enhanced.

[0139] In the present invention, gene introduction means that an exogenous gene is introduced into a host microorganism by means of a vector or the like, or is directly inserted into the genome of the host microorganism.

[0140] In the present invention, enhanced gene expression means that, when a peptide or protein produced by the gene is not present in the host microorganism, the gene is artificially expressed in the host microorganism to give it the activity or function of the peptide or protein; or, when the gene is already present in the host microorganism, the gene is overexpressed or modified so that the activity or function of the peptide or protein produced by the gene is enhanced compared to its endogenous activity or function by using a series of methods, such as switching the endogenous promoter that controls the expression of the gene to a strong constitutive promoter, or increasing the copy number of the gene by further introducing the gene from outside using a vector with strong replicability.

[0141] In the present invention, attenuation of gene expression is a concept that encompasses modifications that weaken the activity or function of a peptide or protein produced by the gene compared to its intrinsic activity or function, by preventing the gene from being expressed or from exhibiting activity or function even if expressed, by mutating, substituting, or deleting some or all of the bases of the gene, or by introducing an inhibitor (e.g., sRNA) that can inhibit gene expression.

[0142] The term "intrinsic activity or function" as used herein means an activity or function possessed by an enzyme, peptide, protein, etc. that a microorganism originally possesses in an unmodified state.

[0143] In the present invention, "modified to enhance an endogenous activity or function" means a state in which an activity or function possessed by a microorganism after manipulation is newly generated or increased compared to the activity possessed by the microorganism before manipulation, such as by introducing a gene exhibiting the activity or function, or increasing the copy number of the gene (e.g., expression using a plasmid into which the gene has been introduced), deleting an inhibitory regulator of the gene expression, or modifying the expression regulatory sequence, for example, by using an improved promoter.

[0144] In the present invention, "modified to weaken the endogenous activity or function" means a state in which the function possessed by the microorganism after manipulation, such as deletion of a gene exhibiting an activity or function, inactivation of the gene (e.g., replacement with a mutant gene), weakening of gene expression (e.g., replacement with a weak promoter, introduction of siRNA, gRNA, sRNA, etc., replacement of the start codon from ATG to GTG, etc.), or inhibition of the function of a peptide expressed by the gene (e.g., addition of a non-competitive inhibitor or competitive inhibitor), is reduced or lost compared to the function possessed by the microorganism before the manipulation.

[0145] In the present invention, "switching" of a gene or promoter means removing the conventional gene or promoter and introducing a different gene (e.g., a mutant gene) or a promoter with different strength. Removing the conventional gene or promoter is a concept that encompasses not only deleting the gene or promoter but also suppressing or reducing its function.

[0146] In the present invention, "overexpression" refers to a level of expression that is higher than the level at which the gene is normally expressed in cells, and includes methods such as replacing the promoter of the gene present on the genome with a stronger promoter, or cloning the gene into an expression vector and transforming it into cells to increase the expression level.

[0147] In the present invention, a "vector" refers to a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. A vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, can integrate into the genome itself. Because plasmids are currently the most commonly used form of vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the present specification. For purposes of the present invention, it is preferable to use a plasmid vector. A typical plasmid vector that can be used for such purposes has a structure that includes (a) an origin of replication that allows efficient replication, with several to several hundred plasmid vectors per host cell, (b) an antibiotic resistance gene that allows selection of host cells transformed with the plasmid vector, and (c) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if a suitable restriction enzyme cleavage site is not present, the vector and foreign DNA can be easily ligated using synthetic oligonucleotide adaptors or linkers according to conventional methods. After ligation, the vector must be transformed into a suitable host cell. Transformation can be easily achieved using the calcium chloride method or electroporation (Neumann, et al., EMBO J., 1:841, 1982), for example.

[0148] The promoter of the vector may be constitutive or inducible and can be further modified to achieve the effects of the present invention. Furthermore, the expression vector contains a selectable marker for selecting host cells containing the vector, and if it is a replicable expression vector, it contains an origin of replication (Ori). The vector can be self-replicating or can be integrated into the host genomic DNA. Preferably, the gene inserted and transferred into the vector is irreversibly integrated into the genome of the host cell, allowing stable and sustained gene expression in the cell for a long period of time.

[0149] A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. This may be a gene and regulatory sequence(s) linked in a way that allows for gene expression when the appropriate molecule (e.g., a transcriptional activator protein) is bound to the regulatory sequence(s). For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a proprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to DNA for a coding sequence if it affects the transcription of the sequence; a ribosomal binding site is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosomal binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous. Linking of these sequences is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0150] As is well known in the art, to increase the expression level of a transformed gene in a host cell, the gene must be operably linked to transcriptional and / or detoxification expression control sequences that function in the selected expression host. Preferably, the expression control sequences and / or the gene are contained within a single recombinant vector that also contains a bacterial selectable marker and origin of replication. If the host cell is a eukaryotic cell, the recombinant vector must also contain an expression marker useful in eukaryotic expression hosts.

[0151] Host cells transformed with the recombinant vectors described above constitute another aspect of the present invention. As used herein, the term "transformation" means introducing DNA into a host so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration.

[0152] Of course, it should be understood that not all vectors function equally well in expressing the DNA sequences of the present invention. Similarly, not all hosts function equally well with the same expression system. However, one skilled in the art can select from a variety of vectors, expression control sequences, and hosts without undue experimental burden and without departing from the scope of the present invention. For example, when selecting a vector, the host must be considered, since the vector must be replicated therein.

[0153] From another aspect, the present invention relates to a method for producing a polyketide glycoside or a phenylpropanoid glycoside, which comprises the following steps: (a) culturing a recombinant microorganism into which a nucleic acid encoding a C-glycosyltransferase mutant of the present invention has been introduced to produce a polyketide glycoside or a phenylpropanoid glycoside; (b) recovering the produced polyketide glycoside or phenylpropanoid glycoside.

[0154] In the present invention, the step (a) may be characterized by adding a precursor of a polyketide glycoside or a phenylpropanoid glycoside and culturing a recombinant microorganism into which a nucleic acid encoding a C-glycosyltransferase mutant has been introduced.

[0155] In the present invention, the recombinant microorganism into which the nucleic acid encoding the C-glycosyltransferase mutant in step (a) has been introduced can be characterized in that the nucleic acid encoding the C-glycosyltransferase mutant has been introduced into a host microorganism capable of producing a precursor of a polyketide glycoside or a phenylpropanoid glycoside, and the host microorganism can be characterized in that an exogenous gene has been introduced or gene expression has been regulated.

[0156] In the present invention, a recombinant microorganism into which a nucleic acid encoding the C-glycosyltransferase mutant has been introduced can have the same characteristics as those described for the recombinant microorganism for producing polyketide glycosides and / or phenylpropanoid glycosides of the present invention.

[0157] In the present invention, step (a) may be characterized by culturing the microorganism by adding ascorbic acid to the culture medium during the culture. In this case, the microorganism may be cultured by adding preferably 0.1 to 1.5 g / L, more preferably 0.2 to 1.0 g / L, of ascorbic acid, but is not limited thereto.

[0158] Unless otherwise specified, the manufacturing method of the present invention may have features equivalent to those described above in the other aspects within the scope of understanding of a person skilled in the art.

[0159] In another aspect, the present invention provides a method for producing a polyketide glycoside and / or a phenylpropanoid glycoside, the method comprising the following steps: (a) reacting a C-glycosyltransferase mutant of the present invention or a microorganism expressing the C-glycosyltransferase mutant with a polyketide and / or a phenylpropanoid to produce a polyketide glycoside and / or a phenylpropanoid glycoside; (b) recovering the produced polyketide glycoside and / or phenylpropanoid glycoside.

[0160] Although specific gene names are described in the present invention, it will be obvious to those skilled in the art that the present invention is not limited to these genes.

[0161] On the other hand, although the gene introduced in the present invention is described by the name of a gene derived from a specific microorganism, the scope of protection of the present invention is not limited to the name of the gene, and it is obvious that if a gene derived from another microorganism with a different gene name is introduced in accordance with the technical features of the present invention within the scope that a person skilled in the art would recognize as having the same function as the gene, the recombinant microorganism also falls within the scope of protection of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

[0162] The present invention will be described in more detail below with reference to specific embodiments. However, the present invention is not limited to the following embodiments, and it is obvious to those skilled in the art that various modifications and alterations can be made within the scope of the present invention.

[0163] (Embodiment 1. Experimental Method) 1-1.Flask culture Flask culture was performed under the following conditions: a colony was inoculated into 10 mL of LB medium supplemented with the appropriate antibiotic concentration and cultured overnight at 37°C. The culture was then subcultured in a 250 mL baffled flask containing 50 mL of R / 2 medium supplemented with 3 g / L yeast extract, 20 g / L glucose (and 0.45 g / L ascorbic acid, if necessary), and cultured at 30°C and 200 rpm. The composition of R / 2 medium (pH 6.8) was as follows (per liter): 2g (NH4)2HPO4, 6.75g KH2PO4, 0.85g citric acid, 0.7g MgSO4·7H2O, and 5ml trace metal solution (TMS) [10g FeSO4·7H2O, 2.25g ZnSO4·7H2O, 1g CuSO4·5H2O, 0.5g MnSO4·5H2O, 0.23g Na2B4O7·10H2O, 2g CaCl2·2H2O, and 0.1g (NH4)6Mo7O]. 24 per liter of 5M HCl). When the OD600 of the culture medium reached 0.6-0.8, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce expression of the foreign gene. After induction, the cells were cultured for 48 hours.

[0164] 1-2. Fed-batch fermentation Fed-batch fermentation was performed in a 6.6 L BioFlo 320 fermenter (Eppendorf) in 1.95LR / 2 medium (pH 6.8) containing 20 g / L glucose, 3 g / L yeast extract, 0.45 g / L ascorbic acid, and the appropriate antibiotic. A colony was inoculated into 10 mL of LB medium supplemented with the appropriate antibiotic concentration and grown overnight at 37°C. The resulting culture was then subcultured in a 250 mL baffled flask containing 50 mL of R / 2 medium supplemented with 3 g / L yeast extract, 20 g / L glucose, and 0.45 g / L ascorbic acid, and grown at 30°C and 200 rpm until an OD600 of approximately 4 was reached. The fermenter was then inoculated, and the pH was maintained at 6.8 by automatic addition of ammonia solution, and the temperature was maintained at 30°C. Oxygen saturation (DO) was maintained at 40% of the air saturation level, and 1vvm [(air volume)·(working volume) -1 (minute) -1 DO was maintained by continuously bubbling air at 1000 kJ / L, increasing the agitation speed, or increasing the concentration of added pure oxygen. IPTG (0.5 mM) was added when the OD reached approximately 20, and the depleted carbon source and other nutrients were automatically added to the fermenter using a pH-stat strategy. The feed solution contained the following components per liter: 650 g glucose, 5 g ascorbic acid, 6 mL TMS, and 8 g MgSO4 7H2O. The feed solution was automatically added to the fermenter when the pH rose above 6.85.

[0165] 1-3. Production volume analysis After cultivation, production analysis was performed under the following conditions. After flask cultivation, 50 mL of culture medium was centrifuged at 4,000 g for 30 minutes, and the supernatant was concentrated by removing salts. An Oasis HLB Cartridge (Water) was used for this. FK was concentrated 1x, KA 30x, dcII 45x, and carminic acid 200x. The concentrated samples were redissolved in an appropriate volume of DMSO and then filtered through a 0.22 μm PTFE filter to remove impurities. The prepared samples were analyzed using an HPLC (1100 series; Agilent) coupled with MS (LC / MSD VL; Agilent). An Eclipse XDB-C18 column was used, with 0.1% formic acid as buffer A and methanol as buffer B. Analysis was performed in ESI negative mode. For a more accurate analysis of carminic acid, LC-MS / MS analysis was performed via an HPLC Triple Quadrupole Mass Spectrometer (LCMS-8050, Shimadzu) (MRM mode).

[0166] For aloesin analysis, LC-MS / MS analysis was performed using an Agilent 6550 iFunnel Q-TOF LC / MS System coupled to an ultra-high performance liquid chromatography (UHPLC; 1290 Infinity II LC System; Agilent). An Eclipse-plus C18 column was used, and buffer A was 0.1% formic acid, and buffer B was acetonitrile supplemented with 0.1% formic acid.

[0167] (Embodiment 2: Investigation of C-glycosyltransferase for carminic acid production) Although the specific pathway for carminic acid production has not yet been elucidated, the carbon skeleton of carminic acid has an anthraquinone-series structure, so we attempted to induce carminic acid production using PKS (Figure 1).

[0168] For this purpose, we used the E. coli BAP1 strain (E. coli BL21(DE3) (Invitrogen)) in which the Bacillus subtilis Sfp gene was introduced into the genome for the activation of exogenous acyl carrier protein (ACP). For this purpose, we utilized the method described in B. Pfeifer et al., Science 2001, 291(5509), 1790-1792 and D. Yang et al., PNAS 2018, 115(40), 9835-9844. To apply the Photorhabdus luminescens Type II PKS, we constructed pDS00-antDEFBG to introduce the P. luminescens antD (ketosynthase), antE (chain-length factor), antF (ACP), antB (phosphopantetheinyl transferase), and antG (malonyl-CoA:ACP malonyltransferase). First, the antDEF gene was PCR-amplified from P. luminescens genomic DNA using the antDE_F and antDEF_R primers and then inserted into the NdeI and EcoRI restriction sites of pDS00 (a plasmid from Novagen, using the same platform as the pET-30a(+) primers, except for the restriction enzyme sequences). The pDS00 plasmid was constructed as follows: a gene fragment containing the T7 promoter, multiple cloning site (MCS), and T7 terminator was amplified from pET-30a(+) using the pET_NheI_DraIII and pET_SpeI_SphI primers, digested with SphI and DraIII, and inserted into the SphI and DraIII sites of the pET-30a(+) plasmid to construct the pDS00 plasmid. Subsequently, antB was amplified from P. luminescens genomic DNA using the antB_F and antB_R primers and inserted into the HindIII site of pDS00 to construct the pDS00-antB plasmid.Next, after digestion with NdeI and EcoRI restriction enzymes, the pDS00-antDEF plasmid was also digested with NdeI and EcoRI restriction enzymes to obtain the antDEF fragment. The two fragments were then combined using Gibson assembly to obtain the pDS00-antDEFB plasmid. The antG gene was then amplified from P. luminescence genomic DNA using the antG_F and antG_R primers and inserted into the NdeI and EcoRI sites of pDS00 to construct the pDS00-antG plasmid. The constructed plasmid was then digested with NheI and SpeI restriction enzymes to obtain the antG fragment, which was then inserted into the NheI site of the pDS00-antDEFB plasmid to construct the pDS00-antDEFBG plasmid.

[0169] [Table 1]

[0170] Next, we constructed the pFK(pDS00-antDEFBG-zhuIJ) plasmid to introduce the ZhuI and ZhuJ cyclases from Streptomyces sp. R1128 for the production of flavokermetic acid (FK). First, using the zhuIJ DNA, codon-optimized for expression in E. coli, we amplified the zhuIJ fragment using the zhuI_F and zhuJ_R primers and inserted it into the NdeI and ECoRI sites of pDS00. The resulting pDS00-zhuIJ was digested with NheI and SpeI restriction enzymes to obtain the zhuIJ fragment, which was then inserted into the NheI site of pDS00-antDEFBG to construct pFK.

[0171] A strain transformed with pDS00-antDEFBG-zhuIJ into BAP1 produced 88 mg / L of FK from glucose. The culture medium was bright red at the beginning of the culture, but over time, it was observed to turn cloudy brown. This is assumed to be due to the conversion of FK to a melanin analogue. To prevent this, 0.45 g / L of ascorbic acid was added to the medium, which increased FK production to 154.9 mg / L.

[0172] [Table 2]

[0173] We predicted that increasing the intracellular concentration of malonyl-CoA would also increase FK production, so we overexpressed the Corynebacterium glutamicum acetyl-CoA carboxylase (encoded by the accBCD1 gene) or knocked down the pabA gene. As a result, FK production increased to 180.3 mg / L in the accBCD1-overexpressing strain (Figure 2).

[0174] Flask culture was initiated by inoculating a colony from an LB agar plate into a test tube containing 10 mL of LB. The appropriate antibiotic concentration was then added and the mixture was incubated overnight at 37°C and 220 rpm. One mL of this seed culture was then inoculated into a 250 mL baffled flask containing 50 mL of R / 2 medium (supplemented with 3 g / L yeast extract and 20 g / L glucose) and incubated at 30°C and 200 rpm. The composition of R / 2 medium (pH 6.8, per liter): 2 g (NH4)2HPO4, 6.75 g KH2PO4, 0.85 g citric acid, 0.8 g MgSO4·7H2O, and 5 mL trace metal solution (TMS). The composition of TMS (per liter, 0.1M HCl base) is as follows: 10g FeSO4·7H2O, 2.25g ZnSO4·7H2O, 1g CuSO4·5H2O, 0.58g MnSO4·5H2O, 0.02g Na2B4O7·10H2O, 2g CaCl2·2H2O, 0.1g (NH4)6Mo7O 24 4H2O. When the OD600 of the culture reached 0.6-0.8, 0.5 mM IPTG was added to induce expression of the exogenous enzyme. The culture was then continued for 48 hours. In the experiments for the production of flavokermetic acid, kermesic acid, dcII, and carminic acid, 0.45 g / L of ascorbic acid was added.

[0175] As a result of the flask culture, a small amount of kermesic acid (KA) was also observed (0.14 mg FK equivalent / L; i.e., mg FK eq / L). This was thought to be due to the endogenous oxidase of E. coli or ZhuIJ, but the conversion efficiency was too low and the enzyme responsible for this reaction has not yet been identified. In the present invention, a biochemical reaction analysis was performed using previously reported literature and compound databases.

[0176] As a result, the gene encoding aklavinone 12-hydroxylase (DnrF) from Streptomyces peucetius was predicted to be the enzyme responsible for the reaction. After amplification using dnrF_F and dnrF_R primers, the gene was inserted into pDS00 at the NdeI and EcoRI sites to construct pDS00-dnrF. Using this plasmid as a base, the dnrF gene was PCR-amplified using pET30a_frag_F and pET30a_frag_R primers, and then cloned into pBBR1-T7 plasmid (Kovach, ME; Phillips, RW; Elzer, PH; Roop, RM, II; Peterson, KM, pBBR1MCS: a broad-host-range cloning vector. Biotechniques 1994, 16(5), 800-802.) using SYPark et al. The fragment (constructed using the method described by et al., bioRxiv, DOI: 10.1101 / 2020.11.27.401000) was amplified by inverse PCR using the pET30a_IV_R and rrnB_IV_F primers, and the two DNA fragments were ligated using Gibson assembly to construct pBBR1-dnrF. After introducing pBBR1-dnrF into the FK-producing strain, flask culture was performed. As a result, 1.20 mg of FK eq / L of KA was produced (Figure 3a).

[0177] [Table 3]

[0178] The production of carminic acid (CA) from FK can be achieved through two biosynthetic pathways, both of which require a monooxygenase and a C-glycosyltransferase. FK can be oxidized to KA or C-glycosylated to dcII. DcUGT2 from D. coccus was found to catalyze the conversion of FK to dcII (or KA to CA) and demonstrated activity in S. cerevisiae (Kannangara et al., Nat Commun 2017, 8). However, because the enzyme must be glycosylated for activity and contains a transmembrane helix and signal peptide, it was predicted that it would be difficult to successfully express in cells such as E. coli. Indeed, DcUGT2 failed to produce dcII when introduced into FK-producing E. coli. To address these DcUGT2 expression problems, we constructed Ntr-DcUGT2, in which the N-terminal signal peptide was removed; Ctr-DcUGT2, in which the C-terminal transmembrane helix was removed; and Ntr-Ctr-DcUGT2, in which both the N-terminal signal peptide and the C-terminal transmembrane helix were removed. We also constructed plasmids carrying the E. coli OmpA signal peptide at the N-terminus of Ntr-DcUGT2 and Ntr-Ctr-DcUGT2, but all of these failed to produce dcII. Therefore, we concluded that DcUGT2 is not active in E. coli.

[0179] Although several cases of O-glycosylation of natural products in E. coli have been reported, very few cases of C-glycosylation have been reported. Therefore, in this study, we selected UDP-glycosyltransferases that were confirmed to perform C-glycosylation in E. coli through biochemical reaction analysis. The eight enzyme candidates selected were: IroB (EnCGT) from E. coli Nissle, UGT708A6 (ZmCGT)C dual / O-glycosyltransferase from Zea mays, UGT708C2 (FeCGT) from Fagopyrum esculentum, MiCGT from Mangifera indica, OsCGT from Oryza sativa, UGT708D1 (GmCGT) from Glycine max, GtUF6CGT1 (GtCGT) from Gentiana triflora, and AvCGT from Aloe vera (Figure 4).

[0180] For the selected enzymes, pCDF-DcCGT, pCDF-MiCGT, pCDF-SfCGT, pCDF-EnCGT, pCDF-OsCGT, pCDF-FeCGT, pCDF-GmCGT, pCDF-AvCGT, pCDF-AvCGT, and pCDF-ZmCGT were constructed. All genes were artificially synthesized except for the iroB gene, which was amplified from E. coli Nissle genomic DNA using the iroB_gib_F and isoB_gib_R primers and inserted into the NdeI site of the pCDFDuet-1 plasmid using Gibson assembly.

[0181] [Table 4]

[0182] Only GtCGT and ZmCGT were able to successfully convert FK to dcII. However, in the case of ZmCGT, the major product was O-glycosylated FK, and only small amounts of dcII were produced. In the case of GtCGT, 0.13 mg CA equivalent / L (mg CA eq / L) of dcII was produced (Figure 5). Because C-glycosylation requires a high level of UDP-glucose, overexpression of galU (encoding UTP-glucose-1-phosphate uridylyltransferase), pgm (encoding phosphoglucomutase), and ndk (encoding nucleoside-diphosphate kinase) resulted in increased dcII production to 0.30 mg CA eq / L (Figure 5). To construct the pBBR1-galU-pgm-ndk plasmid (all three genes were amplified from E. coli BL21(DE3) strain), the galU gene was first amplified with primers galU_gib_F and galU_gib_R and inserted into the EcoRI site of the pBBR1TaC plasmid via Gibson assembly. The pgm gene was amplified with primers pgm_gib_F and pgm_gib_R.

[0183] It was inserted into the KpnI site of the pBBR1TaC-galU plasmid, and the ndk gene was amplified from the ndk_gib_F and ndk_gib_R primers and inserted into the SphI site of the pBBR1-galU-pgm plasmid, thus constructing pBBR1-galU-pgm-ndk.

[0184] [Table 5]

[0185] To improve the activity of GtCGT and DnrF and successfully produce carminic acid, we attempted to create mutations with increased activity through computer simulation. However, because the structure of the enzyme was unknown, we first predicted the protein structure using MODELLER (Webb, B.; Sali, A., Comparative protein structure modeling using MODELLER. Curr Protoc Bioinformatics 2016 54, 5.6.1-5.6.37). We then screened for mutations with increased activity through docking simulations using PyRosetta (Chaudhury, S.; Lyskov, S.; Gray, JJ, PyRosetta: a script-based interface for implementing molecular modeling algorithms using Rosetta. Bioinformatics 2010, 26(5), 689-691). In addition to computer simulation-based predictions, we further selected mutations predicted to increase activity through structural analysis.

[0186] Homology modeling of GtCGT was performed using TCCGT (C-glycosyltransferase from Trollius chinensis; PDB ID 6JTD; protein sequence similarity 35.1%) as a comparison group. Using the calculated GtCGT structural model, computer-based docking simulations (SW: AutoDock Vina) were performed with FK as the ligand. As a result, 239 mutations were calculated, of which 122 mutations showed higher docking scores than the wild-type enzyme (Table 6). Experiments were performed on the top 20 mutations, but 14 mutations predicted to increase activity based on the predicted structure of GtCGT were also tested. The 34 mutations were transformed into the FK strain and cultured in flasks. Six mutations (V93Q, Y193F, L164G, F17G, R322D, and V132A) were identified that showed higher KA production than wild-type GtCGT. Among these, the mutation that showed the highest dcII production was GtCGT V93Q The production was shown to increase by approximately 2.9-fold (Fig. 6c). In this mutation, the Gln93 amino acid was located in the activation site and was determined to directly bind to FK. The Gln93 amino acid forms a hydrogen bond with the hydroxyl group of C6, which is predicted to be in the correct orientation for C-glycosylation of the FK ligand at C2. The Y193F mutation showed the second highest dcII concentration, but to examine the synergistic effect between the two mutations, a double mutation (GtCGT) was constructed. V93Q / Y193F ) was introduced into the FK strain. The double mutation produced 0.74 mg CA eq / L of dcII, a 5.3-fold increase compared to wild-type GtCGT (Figure 6c). In the V93Q mutation, the Tyr193 amino acid forms a hydrogen bond with the carbonyl group at C10, preventing Gln93 from forming a hydrogen bond with the hydroxyl group at C6. Therefore, changing Tyr193 to Phe193 would disrupt the hydrogen bond at C10 and improve ligand binding of FK (Figure 6d).

[0187] [Table 6] *The 20 with the highest docking scores are in bold. Further structure-based selected mutations are in blue. ‡Wild-type GtCGT

[0188] A similar method was used to create a mutant library for DnrF, and the result showed that the mutant with the highest KA production was DnrF. P217K The KA production increased by approximately 2.2-fold (2.68 mg FK eq / L) (Fig. 6a, 6b).

[0189] Mutations for specific sequences were generated in the same manner as previously reported (Zheng, L.; Baumann, U.; Reymond, JL. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res 2004, 32(14), e115.). P217K The plasmid pBBR1-T7 into which V93Q / Y193F The pCDFDuet-1 plasmid containing the introduced fragment was named pdcII. The DnrF_P217K_F and DnrF_P217K_R primers were used to create the P217K mutation in DnrF, the GtCGT_V93Q_F and GtCGT_V93Q_R primers were used to create the V93Q mutation in GtCGT, and the GtCGT_Y193F_F and GtCGT_Y193F_R primers were used to create the Y193F mutation in GtCGT.

[0190] [Table 7]

[0191] GtCGT produced in this invention V93Q / Y193F (GtUF6CGT1 V93Q / Y193FThe protein sequence of the mutated ATP is as follows:

[0192] MGSLTNNDNLHIFLVCFIGQGVVNPMLRLGKAFASKGLLVTLSAPEIVGTEIRKANNLNDDQPIKVGSGMIRFEFFDDGWESVNGSKPFDVWQYINHLDQTGRQKLPIMLKKHEETGTP VSCLILNPLVPWVADVADSLQIPCATLWVQSCASFSAYYHYHHGLVPFPTESEPEIDVQLPGMPLLKYDEVPDFLHPRTPYPFFGTNILGQFKNLSKNFCILMDTFYELEHEIIDNMCK LCPIKPIGPLFKIPKDPSSNGITGNFMKVDDCKEWLDSRPTSTVVYVSVGSVVYLKQEQVTEMAYGILNSEVSFLWVLRPPSKRIGTEPHVLPEEFWEKAGDRGKVVQWSPQEQVLAHP ATVGFLTHCGWNSTQEAISSGVPVITFPQFGDQVTNAKFLVEEFKVGVRLGRGELENRIITRDEVERALREITSGPKAEEVKENALKWKKKAEETVAKGGYSERNLVGFIEEVARKTGTK

[0193] After constructing the DnrF and GtCGT mutants with increased activity as described above, the two mutant enzymes were combined to construct the CA strain. The CA strain was constructed using pFK and pCA (pCDF-dnrF). P217K -GtCGT V93Q / Y193F ) plasmid was transformed into the BAP1 strain. To insert the two genes into one plasmid, the dnrF gene was amplified from pKA by PCR using the dnrF_NcoI_F and dnrF_BamHI_R primers. P217K pCA was constructed by amplifying the pdcII fragment and inserting it into the NcoI and BamHI sites. The constructed CA strain was cultured in a flask, resulting in the production of 22.2 μg / L of carminic acid (Figure 7). The authenticity of the carminic acid produced from glucose was confirmed by LC-MS / MS analysis, as shown in Figure 7.

[0194] To increase carminic acid production, C. glutamicumac BCD1 overexpression, pabA knockdown, and galU-pgm-ndk overexpression were tested individually or in combination, and the production yield of each strain was as follows (Fig. 7a). pabA KD, 25.9μg / L;accBCD1 OE, 74.9μg / L;galU-pgm-ndk OE, 41.0μg / L;accBCD1 OE-galU-pgm-ndk OE, 49.9μg / L;pabA KD-galU-pgm-ndk OE, 57.7μg / L;pabA KD-accBCD1 OE, 57.2μg / L;pabA KD-accBCD1 OE-galU-pgm-ndk OE, 25.2μg / L, and a strain overexpressing accBCD1 (BL21(DE3)harboring pFK, pCA, pACC;pFK:pDS00 derivative containing-antDEFBG from luminiscens and codon optimized zhuIJ from Streptomyces sp.R1128(P T7 -antDEFBG-T7 T -P T7 -zhuIJ-T7 T );pCA:pCDFDuet-1 derivative containing dnrF P217K and GtCGT V93Q / Y193F in different operands(P T7 -dnrF P217K -T7 T -P T7 -GtCGT V93Q / Y193F -T7 T The highest carminic acid concentration of 74.9 μg / L was confirmed in the pACC:pBBR1TaC derivative containing accBC and accD1 from Corynebacterium glutamicum ATCC 13032. Furthermore, fed-batch fermentation of this strain resulted in the production of 0.65 mg / L of carminic acid.

[0195] [Table 8]

[0196] (Embodiment 3: GtCGT V93Q / Y193F aloesin production by Aloesin is a representative cosmetic additive extracted from Aloe vera. It is used as a skin-whitening agent in the cosmetics industry due to its anti-tyrosinase and anti-melanogenesis effects. Its anti-inflammatory and anti-radical properties have also garnered attention as a potential drug or cosmetic ingredient. However, the content of aloesin in plants is very low, necessitating a more efficient production method. Aloesin production has been reported in previous publications (D. Yang et al., Proc. Natl. Acad. Sci. USA 2018, 115(40), 9835-9844). However, a C-glycosyltransferase that converts aloesone to aloesin has not yet been reported (Figure 8). Therefore, in this study, we tested whether the GtCGT mutant developed in Example 2 above could produce aloesin.

[0197] To produce aloesone, we transformed E. coli BL21(DE3) with the following plasmids: pCDF-RpALS, pWAS-anti-pabA, and pBBR1-zwf. Thus, the strain expresses the following genes: RpALS (encoding R. palmatum aloesone synthase), anti-pabA synthesis regulatory sRNA, and zwf (encoding E. coli glucose 6-phosphate 1-dehydrogenase) (see Yang, D.; Kim, WJ; Yoo, SM; Choi, JH; Ha, SH; Lee, MH; Lee, SY. Repurposing type III polyketide synthase as a malonyl-CoA biosensor for metabolic engineering in bacteria. Proc. Natl. Acad. Sci. USA 2018, 115(40), 9835-9844).

[0198] This strain produced 30.9 mg / L of aloesone from glucose. Prior to aloesin production, we attempted to increase aloesone production by introducing RpALS onto a compatible plasmid. To achieve this, RpALS was introduced onto the pRSFDuet-1 plasmid, which contains a high-copy-number RSF replication origin. RpALS was amplified from the previously constructed pCDF-RpALS using primers ALS_NdeI_F and ALS_NdeI_R and then inserted into the NdeI site of pRSFDuet-1 using Gibson assembly. pCDF-RpALS and pRSF-RpALS were then simultaneously transformed into E. coli BL21(DE3) strains already carrying pWAS-anti-pabA and pBBR1-zwf plasmids, and the strains were cultured in flasks. As a result, 102.1 mg / L of aloesone was produced, confirming a significant increase in aloesone production compared to before the improvement.

[0199] [Table 9]

[0200] Then, to test for aloesin production, pCDF-GtCGT or pCDF-GtCGT were transfected onto the BL21(DE3) strain harboring the pWAS-anti-pabA, pRSF-RpALS, and pBBR1-zwf plasmids. V93Q / Y193F After transformation with the plasmid, flask culture was carried out. V93Q / Y193F The strain containing GtCGT produced 0.06 μg / L of aloesin, successfully producing more aloesin than the strain containing GtCGT (Figure 9).

[0201] We attempted to increase the production of aloesin by introducing RpALS in the same way as we did to increase the production of aloesone. V93Q / Y193F Instead of introducing pCDF-RpALS-GtCGT V93Q / Y193F To construct the vector, RpALS was amplified from pCDF-RpALS using pCDFDuet_F and pCDFDuet_R primers and inserted into the NcoI and BamHI sites of the pCA plasmid via Gibson assembly. V93Q / Y193 The four plasmids, pWAS-anti-pabA, pBBR1-zwf, and pWAS-anti-pabA, were transformed into E. coli BL21(DE3), which was then named the ALS strain.

[0202] The ALS strain successfully produced 0.3 μg / L of aloesin from glucose. The authenticity of the produced aloesin was determined by LC-MS / MS, as shown in Figure 9.

[0203] Thus, the present inventors succeeded in producing aloesin through the introduction of GtCGT without even identifying the enzyme. V93Q / Y193F Through the introduction of GtCGT, aloesin production was significantly improved. V93Q / Y193FThe enzyme mutation can be said to be an enzyme capable of producing glycosides across polyketides. In order to construct a mutation that can further enhance aloesin production, the inventors V93Q / Y193F Based on this, we decided to proceed with the creation of additional mutations.

[0204] (Embodiment 4: GtCGT V93Q / Y193F (Increasing aloesin production through further mutation introduction into The present inventors have previously developed GtCGT, which is active against aromatic polyketides. V93Q / Y193F The mutation was further improved to increase the conversion efficiency of aloesone to aloesin. V93Q / Y193F A new substrate, aloesone, was docked onto the structural model, and mutations predicted to form a more stable bond with aloesone and increase enzyme activity were selected as shown in Table 10.

[0205] [Table 10]

[0206] The specific sequence was mutated in the same manner as in Example 2. V93Q / Y193F Using the primer pair in Table 11 below as a template, a plasmid containing a gene mutation was constructed. The constructed plasmid was transformed into the BL21(DE3) strain together with three other plasmids, pWAS-anti-pabA, pRSF-RpALS, and pBBR1-zwf, and the constructed strain was then cultured in a flask under the same conditions as in Example 3. The concentration of the produced aloesin was measured in MRM mode on an HPLC Triple Quadrupole Mass Spectrometer (LCMS-8050, Shimadzu) at the Kaist Biocore Center.

[0207] [Table 11] TIFF0007720917000012.tif127170

[0208] Flask culture revealed that the introduction of the I323S, T50R, T50V, I18P, I95T, Q20M, and I323A mutations increased aloesin production by more than 10-fold. In particular, the GtCGTV93Q / Y193F / I323S mutation increased aloesin production to 7.75 μg / L (Figure 10), a 25.8-fold increase over the existing concentration of 0.3 μg / L.

[0209] In addition to further structure-based mutational exploration, we performed computer-based docking simulations (SW:AutoDock Vina) using the GtCGT structural model with aloezone as a ligand to further enhance enzyme activity. As a result, 15 mutations showed higher docking scores than the wild-type enzyme (Table 12).

[0210] [Table 12]

[0211] The specific sequence was mutated in the same manner as in Example 2. V93Q / Y193F Using the primer pair in Table 13 below as a template, a plasmid containing a gene mutation was constructed. The constructed plasmid was transformed into the BL21(DE3) strain together with three other plasmids, pWAS-anti-pabA, pRSF-RpALS, and pBBR1-zwf, and the constructed strain was then cultured in a flask under the same conditions as in Example 3. The concentration of the produced aloesin was measured in MRM mode on an HPLC Triple Quadrupole Mass Spectrometer (LCMS-8050, Shimadzu) at the Kaist Biocore Center.

[0212] Flask culture revealed that the introduction of the P385A, L194A, and V48G mutations increased aloesin production by more than fivefold. In particular, the GtCGTV93Q / Y193F / P385A mutation increased aloesin production to 4.23 μg / L (Figure 11), a 14.1-fold increase over the existing concentration of 0.3 μg / L.

[0213] [Table 13] TIFF0007720917000015.tif156169

[0214] (Embodiment 5: GtCGT V93Q / Y193F Production of phenylpropanoid glycosides by To test the extendibility of the C-glycosyltransferase developed through the present invention to phenylpropanoid natural products, the present inventors carried out the following experiment.

[0215] Intracellularly expressed GtCGT V93Q / Y193F To confirm the enzyme activity, E. coli BL21(DE3) was transformed with pCDF-GtCGT. V93Q / Y193F The strains transformed with pBBR1-galU-pgm-ndk were cultured in flasks. When cell growth reached an OD60 of 0.6-0.8, 1 mM IPTG was administered. At this time, 70 μM luteolin, 0.5 mM naringenin, or 185.2 μM apigenin were added together and cultured for an additional 36 hours. The amounts of substrate and product were analyzed by LC-MS. Flask cultures were performed in 250 mL baffled flasks containing 50 mL of R / 2 medium (additionally containing 3 g / L yeast extract and 20 g / L glucose) at 30°C and 200 rpm.

[0216] As a result of the cultivation, 15.0 μM vitexin was produced from 185.2 μM apigenin, 51.6 μM naringenin-6-glucoside was produced from 0.5 mM narigenin, and 27.9 μM isoorientin was produced from 70 μM luteolin, which corresponded to conversion rates of 8.1%, 10.3%, and 27.9%, respectively (Figure 10).

[0217] As described above, the glucosyltransferase of the present invention also exhibits the activity of producing various phenylpropanoid C-glucosides, indicating that the enzyme of the present invention is a versatile enzyme capable of producing various polyketides and phenylpropanoid C-glucosides.

[0218] (Embodiment 6: GtCGT V93Q / Y193F Purification and K M , V max measurement) C-glycosyltransferase GtCGT developed through this invention V93Q / Y193F To investigate the properties of GtCGT in more detail, we purified the enzyme and measured the enzyme kinetic parameters. V93Q / Y193F The pCDF-NHis-GtCGT and pCDF-NHis-GtCGTmut plasmids expressing GtCGT were constructed. pCDF-GtCGT and pCDF-GtCGTmut were PCR amplified using GtCGT_N_His_IV_F / GtCGT_N_His_IV_R primers, and then constructed by blunt-end ligation using DpnI and T4 PNK and T4 ligase. The two constructed plasmids were transformed into E. coli BL21(DE3) strain, seed cultured in a test tube containing 10 mL of LB, and grown to an OD of 100 in a flask containing 500 mL of LB. 600The cells were cultured at 37°C until the RI value reached 0.8. For enzyme expression, the cells were treated with 1 mM IPTG and further cultured at 20°C for 16 hours. Cells were harvested by centrifugation and resuspended in 30 mL of lysis buffer (50 mM NaH2PO4, 0.3 M NaCl, 10 mM imidazole, pH 7.5). After sonication, the cells were centrifuged at 10,000 rpm for 40 minutes at 4°C to obtain a supernatant containing water-soluble proteins. The supernatant was passed through TALON resin (Clontech) to purify His-tagged proteins. After removing impurities with wash buffer (50 mM NaH2PO4, 0.3 M NaCl, 20 mM imidazole, pH 7.5), the enzyme was purified by treating the lysis buffer with elution buffer supplemented with 90, 160, 230, or 300 mM imidazole.

[0219] [Table 14]

[0220] The purified enzyme was buffer-exchanged into enzyme storage solution (50 mM HEPES, 20% glycerol, pH 7.5) using Amicon Ultra-15 Centrifugal Filters (regenerated cellulose, 50,000 NMWL; Merck) and filtered. M and V maxTo calculate the γ value, we attempted to convert FK to dcII using purified enzyme. The UDP-Glo Glycosyltransferase Assay Kit (Promega) was used to monitor the extent of the reaction. This kit allows the free UDP generated as a by-product of the reaction to be measured by luminescence. A 200 L enzyme reaction mixture (50 mM HEPES, 0.1 mM UDP-glucose, 5 mM MgCl2, pH 7.5) containing 0.1 M enzyme and various concentrations of FK was reacted at 25°C for 1 hour, after which 25 μL was removed and the luminescence was measured using the kit. The reaction rate and substrate concentration were then applied to the Michaelis-Menten equation, and the γ value was analyzed using OriginPro 2019. V93Q / Y193F K M and V max The values were calculated. V93Q / Y193F K M The value was reduced by 19.5% compared to GtCGT, while GtCGT V93Q / Y193F V max The value increased by 18.2% compared to GtCGT (Figure 13; Table 15). V93Q / Y193F V max / K M The value was improved by 46.8% compared to GtCGT, which is V93Q / Y193F The mutants show improved catalytic efficiency.

[0221] [Table 15]

[0222] (Embodiment 7. Genetic Information) 1. ravC from Streptomyces ravidus (SEQ ID NO: 37) atgtccagtttcagtattgatgatctgaagcgtatcttgcgcgaaggggcaggggcaacggctgagttagacggtgacattttagacgcctcctttgatgatttggggtatgattctttggctcttcttgaaacgggttcgcgcatcggacgtgaatacggtttggaatttgaggatacagctttcgccgacgtggaaacacctcgtgacttggtcggcgtagttaatgctcagttatcggccccggctccgcgtgggtaa

[0223] 2. zhuIJ from Streptomyces sp. R1128 (SEQ ID NO: 38)

[0224] 3. trTT7 from Arabidopsis thaliana (SEQ ID NO: 39)

[0225] 4. ATR2 from Arabidopsis thaliana (SEQ ID NO: 40)

[0226] 5. UrdGT2 (SfCGT) from Streptomyces fradiae TU2717 (SEQ ID NO: 41)

[0227] 6. DcUGT2 (DcCGT) from Dactylopius coccus (SEQ ID NO: 42)

[0228] 7. UGT708A6 (ZmCGT) from Zea mays (SEQ ID NO: 43)

[0229] 8. OsCGT from Oryza sativa (SEQ ID NO: 44)

[0230] 9. UGT708D1 (GmCGT) from Glycine max (SEQ ID NO: 45)

[0231] 10. GtUF6CGT1 (GtCGT) from Gentiana triflora (SEQ ID NO: 46)

[0232] 11. AvCGT from Aloe vera (SEQ ID NO: 47)

[0233] 12. dnrF from Streptomyces peucetius ATCC 29050 (SEQ ID NO: 48)

[0234] 13. antDEF from Photorhabdus luminescens (SEQ ID NO: 49) ATGCGTAA The underlined portion indicates the overlapping portion of the reading frames of antD and antE (ie, the initiation and termination codon sites overlap), and the bold lowercase letters at the end indicate the sequence between antE and antF.

[0235] 14. antB from Photorhabdus luminescens (SEQ ID NO: 50) ATGGACGATATTTCTTTATCATCTGATTTTTTTGATCTTTGGATTATCAAAATCGACGATATTGATTTAGCTTCTATTGAACAGTTAATTCACTGTTCTGATATAGTTCGCCATAACCAAATTTGTTTAGCGGATAGAAGAAAGAGATTTATATTTAGACGGGCTGCATTACGTTATG TTTTGAGTCAATATTTATCTGATTATGAAATCATAACGAATGATAACGGAAACCTTATATATCCACGGAGCAAGACTTCAAATATTATTTTTCACTGAGTGCTTCAGGAAACTATTGTGCCATTGGTTTTAGCTCAAGGGAAATAGGTGTTGATATTGAAGTCACTCCTTCTAAGGTA AAATTTTCAGAAATTATTGAACGTTTTATTAAGGATAAAGATTTGGAATATATGAAAGGTATAATGTTAAAACAACTATCAGGAGTTAGTCTCGGATTTAATAACTATTATCATTTAATGTCATTATATTATTGGGTTAGACTTGAAGCATATATTAAATTATTTGCTTCGACTTTAC ATGAGAAATTATTGGTTAATAACTCTGATTCTGTTAAAGATATGAAAGAATTGGAGGCAAGCACATTATTGATTCATAGTCAGCAATTTGTTGTGCCTTATCTCAAAAGAAAGTCATTTCTACACCAAATATCAAGGAAATAAATTATTCCGAAATTATAAGGAACAAAGATGAGTAA

[0236] 15. antG from Photorhabdus luminescens (SEQ ID NO: 51)

[0237] 16. ScoMCAT from Streptomyces coelicolor (SEQ ID NO: 52) Atgctcgtactcgtcgctcccggccagggcgcccagacgcccggcttcctgactgactggctcgccctccccggtgccgctgaccgcgtcgccgcgtggtcggacgccatcggactcgatctcgcccacttcggcaccaaggccgacgcggacgagatccgagacacgtccgtggcccagccgctgctggtcgccgccggaatcctgtccgccgcggcactcggtacgcagacatctgtcgctgacgcgacgggccccgggttcacccccggcgcggtcgccggacacagcgtcggcgagatcaccgccgccgtcttcgcgggcgtcctcgacgacaccgccgcgctgtccctcgtacgccgtcgcggcctggccatggccgaggccgcggcggtcaccgagaccggcatgtcggcgctgctcgggggcgaccccgaggtgagcgtcgcgcacctggagcggctcggcctgaccccggcgaacgtgaacggcgccggtcagatcgtggcggcgggcaccatggagcagctggccgcgctgaacgaggacaagcccgagggtgtgcgcaaggtcgtcccgctgaaggtggccggcgcgttccacacccgccacatggcccccgccgtggacaagctcgccgaggccgccaaggcgctgacgccggccgacccgaaggtgacgtacgtctccaacaaggacgggcgggccgtcgcctccggcaccgaggtgctggaccggctggtcggccaggtcgccaacccggtgcgctgggacctgtgcatggagacgttcaaggagctgggcgtcaccgcgatcatcgaggtgtgtccgggcggcacgctgaccgggctggccaagcgggcgctgcccggagtgaagacgctggccctgaagacccccgacgacctcgacgcggcccgtgagctcgtcgccgagcacacccaggcctaa

[0238] 17. actVA-orf5 from Streptomyces coelicolor(SEQ ID NO: 53)

[0239] 18. actVB from Streptomyces coelicolor (SEQ ID NO: 54) Atggcagccgaccagggaatgctccgggacgccatggcccgggtgccggccggggtggcgctcgtcaccgcccatgaccgcgggggagtcccgcacggtttcaccgccagttcgttcgtgtccgtctcgatggagccgccactggcactggtctgcctggctcgtacggccaactccttcccggtgttcgacagttgcggcgagttcgcggtgagcgtgctgcgcgaggaccacacggacctggccatgcgcttcgcgcgcaagtccgcggacaagttcgcgggcggggagttcgtccgtaccgcgcggggagcgaccgtgctcgacggagcggtcgcggtcgtcgagtgcacggtccacgagcgctacccggcgggcgaccacatcatcctgctcggcgaggtccagtccgtgcacgtcgaggagaagggcgtaccggcggtctacgtggaccgccggttcgccgccctgtgctcggcggcgggtgcctgcccgtccgccaccgggcggggcgtgcccgcgcatgccggctaa

[0240] 19. pobA from Pseudomonas fluorescens (SEQ ID NO: 55)

[0241] 20. dnrF P217K from Streptomyces peucetius (final dnrF mutation) (SEQ ID NO: 56)

[0242] 21. GtCGT V93Q / Y193F from Gentiana triflora (final GtCGT mutation) (SEQ ID NO: 57) a

[0243] 22. ALS from Rheum palmatum (SEQ ID NO: 58)

[0244] 23. accBC from Corynebacterium glutamicum (SEQ ID NO: 59)

[0245] 24. accD1 from Corynebacterium glutamicum (SEQ ID NO: 60)

[0246] 25. GtCGT V93Q / Y193F (GtUF6CGT1 V93Q / Y193F ) variant (SEQ ID NO: 61) MGSLTNNDNLHIFLVCFIGQGVVNPMLRLGKAFASKGLLVTLSAPEIVGTEIRKANNLNDDQPIKVGSGMIRFEFFDDGWESVNGSKPFDVWQYINHLDQTGRQKLPIMLKKHEETGTP VSCLILNPLVPWVADVADSLQIPCATLWVQSCASFSAYYHYHHGLVPFPTESEPEIDVQLPGMPLLKYDEVPDFLHPRTPYPFFGTNILGQFKNLSKNFCILMDTFYELEHEIIDNMCK LCPIKPIGPLFKIPKDPSSNGITGNFMKVDDCKEWLDSRPTSTVVYVSVGSVVYLKQEQVTEMAYGILNSEVSFLWVLRPPSKRIGTEPHVLPEEFWEKAGDRGKVVQWSPQEQVLAHP ATVGFLTHCGWNSTQEAISSGVPVITFPQFGDQVTNAKFLVEEFKVGVRLGRGELENRIITRDEVERALREITSGPKAEEVKENALKWKKKAEETVAKGGYSERNLVGFIEEVARKTGTK

[0247] 26. zhuIJ - Codon optimization for E.coli (SEQ ID NO: 62) [Explanation of symbols]

[0248] FK: Flavokermesic acid KA: Kermesic acid CA: Carminic acid [Industrial Applicability]

[0249] The C-glycosyltransferase mutants of the present invention have improved glycosidic bond formation ability compared to wild-type C-glycosyltransferases, and can enhance the efficiency of glycoside production of polyketides and related natural products, particularly type I, II, and III polyketides, nonribosomal peptides, phenylpropanoids, and other aromatic natural products. Therefore, the C-glycosyltransferase mutants of the present invention may be useful in producing drugs, food additives, nutritional supplements, and the like, which contain increased amounts of C-glycoside compounds as components through the production of polyketide glycosides of natural products.

Claims

1. A C-glycosyltransferase mutant represented by SEQ ID NO: 1, which comprises the mutations V93Q and Y193F and has increased catalytic efficiency, for use in the production of carminic acid.

2. A C-glycosyltransferase mutant for use in the production of aloesin, which C-glycosyltransferase represented by SEQ ID NO: 1 comprises mutations V93Q and Y193F, and mutations of one or more amino acids selected from the group consisting of the following, and has increased catalytic efficiency: I323S, T50R, T50V, I18P, I95T, Q20M, I323A, P385A, L194A, and V48G.

3. A nucleic acid encoding the variant of claim 1 or 2.

4. A recombinant microorganism into which the nucleic acid of claim 3 has been introduced.

5. The recombinant microorganism according to claim 4, wherein expression of a gene encoding UTP-glucose-1-phosphate uridyltransferase, phosphoglucomutase, and / or nucleoside diphosphate kinase is enhanced.

6. The recombinant microorganism according to claim 4, wherein a polyketide synthase or a phenylpropanoid synthase is further introduced into the recombinant microorganism.

7. The recombinant microorganism according to claim 4, characterized in that the expression of the pabA gene is attenuated.

8. (i) a gene encoding a type II polyketide biosynthetic enzyme; and (ii) a gene encoding 4'-phosphopantetheinyl transferase; and (iii) a gene encoding a cyclase; and (iv) a gene encoding acetyl-CoA carboxylase; and (v) a gene encoding aklavinone 12-hydroxylase.

9. 9. The recombinant microorganism according to claim 8, wherein the gene encoding the type II polyketide biosynthetic enzyme is any one or more genes selected from the group consisting of antD (ketosynthase), antE (chain-length factor), antF (ACP), antB (phosphopantetheinyl transferase), and antG (malonyl-CoA:ACP malonyltransferase), or a combination thereof.

10. The recombinant microorganism described in claim 8, characterized in that the aklavineone 12-hydroxylase contains a mutation (P217K) from proline to lysine at the 217th amino acid in the amino acid sequence shown in SEQ ID NO:

2.

11. The type II polyketide biosynthetic enzyme is derived from P. luminescens, The 4'-phosphopantetheinyl transferase is derived from Bacillus subtilis or P. luminescens; The cyclase is derived from Streptomyces sp. the acetyl-CoA carboxylase is derived from Corynebacterium glutamicum, and / or The recombinant microorganism according to claim 8, wherein the aklavineone 12-hydroxylase is derived from Streptomyces peucetius.

12. 6. The recombinant microorganism of claim 5, wherein (i) a gene encoding aloezone synthase has been introduced into the microorganism for use in producing aloesin.

13. 13. The recombinant microorganism according to claim 12, wherein the aloezone synthase is derived from R. palmatum.

14. A method for producing carminic acid or aloesin, comprising the steps of: (a) culturing the recombinant microorganism of claim 4 to produce carminic acid or aloesin; (b) recovering the carminic acid or aloesin produced.

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