Synthetic gene cluster of enfumafungin antibiotic and synthesis method therefor

Abstract

The present invention relates to a synthetic gene cluster of an enfumafungin antibiotic and a synthetic method therefor. Specifically, in the present invention, key functional genes in the linking of a β-D-glucopyranose at position C3 of a fernane-type framework, the oxidation at position C2 into α-OH, the oxidative cleavage of ring E at C19-C20, and the acetylation of hydroxyl at position C2 during the biosynthesis of an enfumafungin antibiotic, i.e. fuscoatroside, are isolated, wherein the genes are named fsoA, fsoD, fsoE and fsoF, respectively. The present invention also provides encoding polypeptides thereof. Provided in the present invention are an artificial fusion enzyme gene, i.e. efuA(TC)fsoA(GT); and on the basis of the artificial fusion enzyme gene, the heterologous expression of four genes, i.e. efuA(TC)fsoA(GT), fsoD, fsoE and fsoF, can synthesize an enfumafungin precursor (13). The present invention clarifies an FsoE-mediated C-C bond breaking function of a P450 enzyme, and provides a key catalytically active residue of FsoE. The present invention reports the biosynthetic pathway of such compound for the first time, and establishes an important foundation for the green and efficient synthesis of the compound.

Description

Synthetic gene cluster and synthesis method of animafenjin antibiotics Technical Field

[0001] The present invention belongs to the field of genetic engineering and biosynthesis, and particularly relates to the biosynthesis of a class of anemaphene gold antibiotics. Background Art

[0002] Enfumafungin antibiotics are a class of fungal triterpenoids with a unique structure, based on a fernane-type triterpene skeleton, with C3β-OH linked to β-D-pyranose, C2 oxidized to α-OH, and E ring C19-C20 cleaved. Representative compounds include enfumafungin. [1] , fuscoatroside [2] ,kolokoside A [3] , WF11605 [4] Anmafenjin antibiotics are not only novel in structure but also have outstanding antifungal activity. Among them, ibrexafungerp, developed with anmafenjin as a precursor, has obtained clinical drug approval from the US FDA for oral treatment of invasive gynecological candidiasis. [5] . At present, airefungin is mainly obtained by fermenting the original strain to obtain anafenacin, which is then hydrolyzed into aglycone and then chemically modified. There are problems such as long fermentation cycle, low yield, and complex process. In addition, due to the complex structure of anafenacin antibiotics and the presence of multiple chiral centers, their chemical total synthesis has not been reported so far. Therefore, elucidating the biosynthetic mechanism of anafenacin antibiotics can not only solve the drug source problem of this type of compound, but also lay the foundation for the use of biosynthesis technology to discover anafenacin derivatives with better activity.

[0003] In 2018, researchers first discovered a potential biosynthetic gene cluster for phenoxyethanol from the genome of Hormonema carpetanum ATCC74360 (Figure 8). There were 12 genes in total, including three P450 enzyme genes, but the distance between genes efuF and efuA was large, suggesting that they may not belong to the same gene cluster. Subsequently, researchers knocked out the fusion gene efuA of terpene cyclase and glycosyltransferase in the gene cluster and found that the mutant strain of efuA no longer produced phenoxyethanol, thus identifying the biosynthetic gene cluster of phenoxyethanol for the first time. [6] On this basis, the researchers speculated on the biosynthetic pathway of animafenine (Figure 9) and proposed a multi-enzyme-mediated E-ring cleavage process. First, C19 is oxidized by P450 enzymes to form a C19 carbonyl group, followed by a Baeyer-Villiger (BV enzyme) reaction to form a 6-membered lactone ring. The lactone ring is then hydrolyzed, dehydrated, and reduced to form the final product. [6]. There are three doubts about the above-specified BV ring cleavage pathway: First, the key C20-C21 double bond intermediate product is a stable product, but it has not been found in nature; second, the BV enzyme, esterase, dehydratase and reductase required for this pathway cannot be found in the gene cluster; finally, the chirality of the C21 position before and after cleavage is highly conserved, suggesting that the hydrogen at C21 is likely not removed during the reaction. Based on this, we believe that the cleavage of the E ring C19-C20 is likely to involve other unknown carbon-carbon cleavage mechanisms. In summary, although the gene cluster of animafenkin has been reported, the functions of all genes in the gene cluster, including efuA, have not been elucidated, and the biosynthetic mechanism of animafenkin is still unclear.

[0004] In previous studies, the inventors obtained a brown-black humicola fungus Humicola fuscoatra NRRL 22980 from the U.S. Agricultural Research Service (NRRL), which can produce a high yield of the fuscoatroside antibiotic, which laid a material foundation for the inventors to study the biosynthesis mechanism of fuscoatroside antibiotics. Summary of the Invention

[0005] The inventors obtained a fungal strain, Humicola fuscoatra NRRL22980, that produces the phenoxyl antibiotic fuscoatroside from the U.S. Agricultural Research Service (NRRL) and isolated the fuscoatroside biosynthetic gene cluster from this strain. This gene cluster comprises four genes (fsoA, fsoD, fsoE, and fsoF), which are capable of synthesizing fuscoatroside in a heterologous host, Aspergillus oryzae. FsoA, a fusion enzyme containing a terpene cyclase (TC) domain and a glycosyltransferase (GT) domain, catalyzes the formation of a fernane backbone with a β-D-glucopyranosyl group attached to the C-3 position from 2,3(S)-epoxysqualene. The P450 enzyme FsoD is responsible for oxidation at the C2 position to form a C2α hydroxyl group. The P450 enzyme FsoE catalyzes the cleavage of the C19-C20 group to form an oxidized carboxyl group and a reduced methyl group. The acyltransferase FsoF is responsible for acetylation of the C2α hydroxyl group. This is also the first time in this field that the gene responsible for the anafenatin antibiotic fuscoatroside has been identified, that is, only four genes (fsoA, fsoD, fsoE, fsoF) are needed to generate the final C19-C20 cleavage product fuscoatroside, which is significantly different from the biosynthetic pathway of anafenatin antibiotics speculated in the literature.

[0006] In addition, the inventors fused the GT domain in the fusion enzyme FsoA with the TC domain in the reported anemafen financial synthase EfuA to form an artificial fusion enzyme gene efuA (TC) fsoA (GT) , then efuA (TC) fsoA (GT) When expressed simultaneously with three post-modification genes (fsoD, fsoE, and fsoF) in a heterologous host of Aspergillus oryzae, the animaphene gold precursor compound 13 can be synthesized.

[0007] The inventors used substrate feeding experiments to further clarify the function of the P450 oxidase FsoE and found that it can independently catalyze the cleavage of the E-ring C19-C20 position to form a carboxyl group on the left and a methyl group on the right. This phenomenon has never been observed in all reported P450 enzymes. In order to explore the catalytic mechanism of the P450 oxidase FsoE, the inventors performed homology modeling of FsoE based on AlphaFold2 and AlphaFill (where heme is covalently linked to the conserved residue C517), and then used AutoDock Vina to dock compound 10 into the protein model of FsoE, finding the key residues related to catalytic E-ring cleavage (R315, F148, F259, F337, W339, F557, N344, Y143). Based on the docking results, we performed point mutations on these residues and studied the catalytic function of the key residues through feeding experiments. Results showed that mutations of N344 and R315 to A completely abolished FsoE's enzymatic activity, suggesting that R315 and N344 may play a key role by forming hydrogen bonds with the substrate's C3 hydroxyl group and C19 keto group. Furthermore, mutations of two nonpolar residues, W339 and F337, also completely abolished FsoE's catalytic activity, suggesting that these active sites may bind substrates through hydrophobic interactions. Furthermore, mutations of four residues near the E-loop (F557A, F259A, F148A, and Y143A) still produced the C19 carbonyl product 10, with F557A nearly converting substrate 9 to compound 10. However, all four mutants failed to produce the E-loop cleavage product, indicating that F557, F259, F148, and Y143 play a key role in catalyzing C-C bond cleavage to form the ring-opening product 11. Furthermore, when Y at position 143 was mutated to F, the results showed that the Y143F mutant could only convert substrate 9 to compound 10 but not compound 11, indicating that the hydroxyl group at Y143 is important for C-C bond cleavage. These results advance our understanding of FsoE and provide valuable insights into its development and utilization in C-C bond cleavage.

[0008] The first aspect of the present invention provides an isolated or synthetic polypeptide selected from:

[0009] (a) an FsoA polypeptide comprising the polypeptide sequence shown in SEQ ID NO: 1 or a polypeptide sequence having at least 70% sequence identity thereto, preferably 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity thereto;

[0010] (b) an FsoD polypeptide comprising the polypeptide sequence shown in SEQ ID NO: 2 or a polypeptide sequence having at least 70% sequence identity thereto, preferably 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity thereto;

[0011] (c) an FsoE polypeptide comprising the polypeptide sequence shown in SEQ ID NO: 3 or a polypeptide sequence having at least 70% sequence identity thereto, preferably 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity thereto;

[0012] (d) an FsoF polypeptide comprising the polypeptide sequence shown in SEQ ID NO: 4 or a polypeptide sequence having at least 70% sequence identity thereto, preferably 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity thereto; and

[0013] (e)EfuA (TC) FsoA (GT) A polypeptide comprising the polypeptide sequence shown in SEQ ID NO: 5 or a polypeptide sequence having at least 70% sequence identity thereto, preferably 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity thereto.

[0014] In one embodiment, the FsoA polypeptide, FsoD polypeptide, FsoE polypeptide, and FsoF polypeptide sequences are derived from Humicola fuscoatra NRRL 22980.

[0015] In one embodiment, the EfuA (TC) FsoA (GT) The GT domain of the polypeptide is FsoA (GT) Source: Humicola fuscoatra NRRL 22980, and the EfuA (TC) FsoA (GT) The TC domain of the polypeptide is EfuA (TC) Derived from the fungus Hormonema carpetanum ATCC 74360.

[0016] The second aspect of the present invention provides a polynucleotide encoding the polypeptide of the first aspect. In a preferred embodiment, the polynucleotide comprises a sequence selected from the following:

[0017] (i) the nucleic acid sequence of SEQ ID NO: 6, or a nucleic acid sequence having at least 70% sequence identity thereto, preferably 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity thereto;

[0018] (ii) the nucleic acid sequence of SEQ ID NO: 7, or a nucleic acid sequence having at least 70% sequence identity thereto, preferably 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity thereto;

[0019] (iii) the nucleic acid sequence of SEQ ID NO: 8, or a nucleic acid sequence having at least 70% sequence identity thereto, preferably 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity thereto;

[0020] (iv) the nucleic acid sequence shown in SEQ ID NO: 9, or a nucleic acid sequence having at least 70% sequence identity thereto, preferably 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity thereto; and

[0021] (v) the nucleotide sequence of SEQ ID NO: 10, or a nucleic acid sequence having at least 70% sequence identity thereto, preferably 80%, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity thereto.

[0022] "Percent identity" refers to the degree to which two optimally aligned DNA or protein segments are invariant throughout the alignment window, e.g., nucleotide or amino acid sequences. The "identity score" for an aligned segment of a test sequence and a reference sequence is the number of identical components shared by the two aligned segment sequences over the alignment window divided by the total number of sequence components in the reference segment, the total number being the lesser of the entire test sequence or the entire reference sequence. "Percent identity" ("% identity") is the identity score multiplied by 100.

[0023] The third aspect of the present invention provides an expression cassette comprising the polynucleotide according to the second aspect of the present invention.

[0024] The fourth aspect of the present invention provides a vector, such as an expression vector, comprising the polynucleotide according to the second aspect of the present invention or the expression cassette according to the third aspect.

[0025] The fifth aspect of the present invention provides a host cell comprising the polynucleotide according to the second aspect of the present invention, the expression cassette according to the third aspect or the vector according to the fourth aspect.

[0026] The gene and gene product of the enzyme of the present invention can be expressed in heterologous host cells, for example bacterial cells, fungal cells, for example yeast cells. The heterologous host cells used to express polynucleotide molecules of the present invention can be microbial hosts that are present in fungi or bacteria families and grow in wide temperature, pH value and solvent tolerance ranges. For example, it is expected that any bacterium, yeast and filamentous fungi can be suitable hosts for expressing nucleic acid molecules of the present invention.Examples of host strains include, but are not limited to, bacterial, fungal, or yeast species such as Humicola, Pichia, Aspergillus, Trichoderma, Saccharomyces, Phaffia, Kluyveromyces, Yarrowia, Candida, Hansenula, Salmonella, Bacillus, us), Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium terium), Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methyl Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella, and Myxococcus species.

[0027] In one embodiment, the host cell is a fungal cell.

[0028] In a preferred embodiment, the host cell is a brown-black humicola, such as a Humicola fuscoatra cell. In another preferred embodiment, the cell is an Aspergillus oryzae cell.

[0029] In one embodiment, the host cell is a bacterial cell, such as an E. coli cell.

[0030] Vectors that can be used to transform the above-mentioned host cells are well known in the art. Generally, the vector comprises sequences that direct the transcription and translation of the relevant genes, a selectable marker, and sequences that allow autonomous replication or chromosomal integration. Suitable vectors comprise a 5' region of the gene containing a transcription initiation control and a 3' region of the DNA fragment that controls transcription termination.

[0031] A sixth aspect of the present invention provides a method for the biosynthesis of fuscoatroside, an anabolic antibiotic, or a precursor thereof, comprising contacting one or more of the FsoA, FsoD, FsoE, and FsoF polypeptides with 2,3(S)-epoxysqualene and uridine diphosphate glucose (UDPG) substrates.

[0032] In one embodiment, the synthesis method comprises: expressing one or more of the FsoA, FsoD, FsoE, and FsoF polypeptides in a host cell, such that the polypeptide catalyzes the synthesis of fuscoatroside or its precursor from a substrate; and isolating fuscoatroside or its precursor from the host cell. In one embodiment, the host cell contains 2,3(S)-epoxysqualene and uridine diphosphate glucose (UDPG) substrate. In one embodiment, the host cell is an Aspergillus oryzae host cell.

[0033] In one embodiment, the synthetic method comprises simultaneously expressing fsoA, fsoD, fsoE, and fsoF in an Aspergillus oryzae host.

[0034] In one embodiment, the synthetic method comprises expressing fsoA alone in an Aspergillus oryzae host.

[0035] In one embodiment, the synthetic method comprises simultaneously expressing fsoA and fsoD in an Aspergillus oryzae host.

[0036] In one embodiment, the synthetic method comprises simultaneously expressing fsoA and fsoE in an Aspergillus oryzae host.

[0037] In one embodiment, the synthetic method comprises simultaneously expressing fsoA, fsoD, and fsoF in an Aspergillus oryzae host.

[0038] In one embodiment, the synthetic method comprises simultaneously expressing fsoA, fsoD, and fsoE in an Aspergillus oryzae host.

[0039] The seventh aspect of the present invention provides a biosynthetic method of an anergyne gold precursor compound 13 or its precursor, which comprises the steps of: (TC) FsoA (GT) One or more of the FsoD, FsoE, and FsoF polypeptides are contacted with 2,3(S)-epoxysqualene and uridine diphosphate glucose (UDPG) substrate.

[0040] In one embodiment, the synthesis method comprises: expressing EfuA in a host cell (TC) FsoA (GT) , FsoD, FsoE and FsoF polypeptides, so that it catalyzes the substrate to synthesize compound 13; and isolates compound 13 from the host cell. The structure of compound 13 is as follows:

[0041] In one embodiment, the host cell contains 2,3(S)-epoxysqualene and uridine diphosphate glucose (UDPG) substrate. In one embodiment, the host cell is an Aspergillus oryzae host cell.

[0042] In one embodiment, the synthetic method comprises simultaneously expressing EfuA in an Aspergillus oryzae host. (TC) FsoA (GT) , FsoD, FsoE and FsoF polypeptides.

[0043] In the above aspects, expression of the polypeptide is achieved by introducing a polynucleotide encoding the polypeptide into a host cell, such as an Aspergillus oryzae host cell.

[0044] The eighth aspect of the present invention provides a polypeptide that catalyzes the cleavage of the E-ring C19-C20 position of a fernane-type compound, which comprises amino acid residues corresponding to R315, F148, F259, F337, W339, F557, N344 and Y143 of the amino acid sequence shown in SEQ ID NO:3.

[0045] The ninth aspect of the present invention provides a method for catalyzing the cleavage of C19-C20 of the E-ring of a fernane-type compound, comprising contacting the FsoE polypeptide of the first aspect of the present invention or the polypeptide of the eighth aspect with a fernane-type compound.

[0046] In one embodiment, the method comprises expressing the FsoE polypeptide of the first aspect or the polypeptide of the eighth aspect of the present invention in a host cell, so that it catalyzes the cleavage of the E-ring C19-C20 of the fernane-type compound.

[0047] In one embodiment, the host cell is an Aspergillus oryzae host cell.

[0048] In one embodiment, the fernane-type compound is linked to a structure;

[0049] In a specific embodiment, the fernane-type compounds are 9 and 10, and their structures are shown in the following formula:

[0050] The tenth aspect of the present invention provides the key catalytic site for P450 enzyme FsoE-mediated CC bond cleavage, which is the amino acid residues corresponding to R315, F148, F259, F337, W339, F557, N344 and Y143 of the amino acid sequence shown in SEQ ID NO:3.

[0051] The eleventh aspect of the present invention provides the use of the polypeptide described in the first or eighth aspect of the present invention, the polynucleotide described in the second aspect, the expression cassette described in the third aspect, the vector described in the fourth aspect or the cell described in the fifth aspect in the synthesis of anemaquine antibiotics.

[0052] The twelfth aspect of the present invention provides a kit comprising the polypeptide described in the first or eighth aspect of the present invention, the polynucleotide described in the second aspect, the expression cassette described in the third aspect, the vector described in the fourth aspect, or the cell described in the fifth aspect.

[0053] The technical effects of the present invention are as follows: 1) The present invention discovered the synthetic genes fsoA, fsoD, fsoE and fsoF for synthesizing a class of anthracene antibiotics represented by fuscoatroside from Humicola fuscoatra NRRL 22980; 2) When fsoA, fsoD, fsoE and fsoF are simultaneously expressed in Aspergillus oryzae, they can utilize Aspergillus oryzae's own 2,3(S)-epoxysqualene and uridine diphosphate glucose to synthesize compound fuscoatroside (1); 3) When fsoA is expressed alone in Aspergillus oryzae, it can utilize Aspergillus oryzae's own 2,3(S)-epoxysqualene and uridine diphosphate glucose to synthesize compounds 2 and 3; 4 ) When fsoA and fsoD are expressed simultaneously in Aspergillus oryzae, compounds 4 and 5 can be produced; 5) When fsoA and fsoE are expressed simultaneously in Aspergillus oryzae, compounds 6 and 7 can be produced; 6) When fsoA, fsoD and fsoE are expressed simultaneously in Aspergillus oryzae, compounds 8, 9, 10 and 11 can be produced; 7) When fsoA, fsoD and fsoF are expressed simultaneously in Aspergillus oryzae, compound 12 can be produced; 8) The present invention fuses the GT domain in the fusion enzyme FsoA with the TC domain in the reported animafen financial synthase EfuA to form an artificial fusion enzyme gene efuA(TC) fsoA (GT) 9)efuA (TC) fsoA (GT) When fsoD, fsoE, and fsoF are simultaneously expressed in a heterologous host of Aspergillus oryzae, they can utilize Aspergillus oryzae's own 2,3(S)-epoxysqualene and uridine diphosphate glucose to synthesize anthracene precursor (13); 10) The present invention uses an Aspergillus oryzae strain expressing fsoE alone to conduct in vivo feeding experiments with compound 9 or 10, and can obtain compound 12 with E ring cleavage respectively. 11) The present invention uses AlphaFold2, AlphaFill, and AutoDock Vina software to perform homology modeling and molecular docking on fsoE, and finds the key amino acids (R315, F148, F259, F337, W339, F557, N344, Y143) responsible for FsoE catalyzing CC bond cleavage, and uses substrate 9 to conduct feeding experiments, revealing the role of key amino acids in catalysis. 12) The present invention has developed a biosynthetic method for anthracene gold antibiotics, which has the advantages of simple process, high stereoselectivity, and low environmental pollution, and has become an important way to produce anthracene gold antibiotics with good biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG1 shows the structural formulas of representative enfumafungin antibiotics, including enfumafungin, fuscoatroside, kolokoside A, and WF11605.

[0055] Figure 2 shows the biosynthetic genes for fuscoatroside compounds and amphetamine precursor (13). Figure 2A shows the fuscoatroside biosynthetic gene cluster from Humicola fuscoatra NRRL 22980 identified in the present invention; Figure 2B shows the specific fusion scheme of the core artificial fusion enzyme gene and the structure of the amphetamine precursor compound (13).

[0056] Figure 3 shows metabolite analysis of the fuscoatroside gene cluster heterologously expressed in Aspergillus oryzae. Figure 3A shows the LC-MS analysis of an extract from an Aspergillus oryzae strain expressing the entire fuscoatroside gene cluster at once; Figure 3B shows the LC-MS analysis of an extract from an Aspergillus oryzae strain expressing individual genes in the fuscoatroside gene cluster in a stepwise manner.

[0057] Figure 4 shows the possible biosynthetic pathway of the anthracene antibiotic fuscoatroside.

[0058] Figure 5 shows the biosynthetic pathway of the animafen gold precursor (13). Figure 5A shows the simultaneous expression of efuA (TC) fsoA (GT) , fsoD, fsoE, fsoF ELSD detection spectrum of Aspergillus oryzae strain extract. Figure 5B shows the biosynthesis process of animafen gold precursor (13).

[0059] Figure 6 shows the detection profiles of compounds 9 and 10, respectively, after being cultured in an Aspergillus oryzae strain expressing only the fsoE gene; Figure 6A is an in vivo HPLC profile of the reaction of an Aspergillus oryzae strain expressing only the fsoE gene with substrates 9 and 10, respectively, with a detection wavelength of UV 208 nm; Figure 6B shows the possible mechanism of FsoE-catalyzed E-ring cleavage.

[0060] Figure 7 shows the search for key amino acid residues for FsoE-mediated C-C bond cleavage. Figure 7A shows the predicted location of substrate 10 in the hydrophobic active pocket and the identification of amino acid residues surrounding the pocket; Figure 7B shows the results of the experiment in which compound 9 was cultured in Aspergillus oryzae containing an FsoE point mutant.

[0061] Figure 8 shows the prior art literature [6] The potential anthracene biosynthetic gene clusters and the predicted functions of each gene were analyzed.

[0062] Figure 9 shows the prior art literature [6] The putative biosynthetic pathway of anthracene. DETAILED DESCRIPTION

[0063] The term "fernane-type triterpene skeleton" refers to a class of compounds found in nature, whose core consists of four six-membered rings and one five-membered ring. Rings A and D are in a chair conformation, rings B and C are in a twist-boat conformation, and the A / B, C / D, and D / E rings are all trans-connected. Fernane-type triterpenes are formed by the cyclization of squalene or 2,3(S)-epoxysqualene as precursors. Due to different carbon cation rearrangements and deprotonation during the cyclization process, fernane-type triterpene skeletons with different double bond positions are formed. Currently, the fernane skeletons found in fungi are mainly formed by the cyclization of 2,3(S)-epoxysqualene as a precursor, including three skeletons with different double bond positions: isomotiol, motiol, and fernenol (see the formula below).

[0064] The term "enfumafungin antibiotics" refers to a class of fungal triterpenes with a unique structure, based on a fernane-type triterpene skeleton, with the C3β-OH group linked to β-D-pyranose glucose, oxidation of the C2 position to an α-OH group, and cleavage of the E ring between C19 and C20. Currently, representative enfumafungin antibiotics found in fungi include enfumafungin, fuscoatroside, kolokoside A, and WF11605 (Figure 1).

[0065] The FsoA protein provided by the present invention is a fusion enzyme containing a terpene cyclase (TC) conserved domain and a glycosyltransferase (GT) conserved domain; the FsoD protein and the FsoE protein are P450 oxidases; and the FsoF protein is an acetyltransferase.

[0066] In one embodiment, when the four proteins are simultaneously expressed in Aspergillus oryzae, the four proteins can synthesize fuscoatroside (1) using Aspergillus oryzae's own 2,3(S)-epoxysqualene and uridine diphosphate glucose as raw materials.

[0067] In another specific embodiment, when fsoA is expressed in Aspergillus oryzae, the obtained strain is capable of producing Compound 2 and Compound 3.

[0068] In another specific embodiment, when fsoA and fsoD are simultaneously expressed in Aspergillus oryzae, the obtained strain is capable of producing Compound 4 and Compound 5.

[0069] In another specific embodiment, when fsoA and fsoE are simultaneously expressed in Aspergillus oryzae, the obtained strain is capable of producing Compound 6 and Compound 7.

[0070] In another specific embodiment, when fsoA, fsoD and fsoE are simultaneously expressed in Aspergillus oryzae, the obtained strain is capable of producing Compound 8, Compound 9, Compound 10 and Compound 11.

[0071] In another specific embodiment, when fsoA, fsoD and fsoF are simultaneously expressed in Aspergillus oryzae, the resulting strain is capable of producing Compound 12.

[0072] In a specific embodiment, the EfuA provided by the present invention is (TC) FsoA (GT) The protein is formed by artificial fusion, which is formed by connecting the GT domain in the fusion enzyme FsoA and the TC domain in the reported anthracene financial synthase EfuA through a head-to-tail connection.

[0073] In a specific embodiment, efuA (TC) fsoA (GT)When the four proteins fsoD, fsoE, and fsoF are simultaneously expressed in Aspergillus oryzae, the four proteins can synthesize the precursor compound of anemaphene gold using 2,3(S)-epoxysqualene and uridine diphosphate glucose as raw materials of Aspergillus oryzae itself (13).

[0074] In one embodiment of the present invention, an in vivo feeding experiment was conducted using an Aspergillus oryzae strain expressing fsoE alone and compound 9 or 10 to obtain compound 12 with E-ring cleavage.

[0075] In one embodiment of the present invention, homology modeling and molecular docking of fsoE were performed using AlphaFold2, AlphaFill, and AutoDock Vina software, and the key amino acids (R315, F148, F259, F337, W339, F557, N344, Y143) responsible for FsoE catalyzing CC bond cleavage were found. Feeding experiments were performed using substrate 9, revealing the role of key amino acids in catalysis.

[0076] The present invention is understood by the following examples, however, it is to be understood that these examples do not limit the present invention. Changes of the present invention now known or further developed are considered to fall within the scope of the present invention described herein and claimed below.

[0077] Example 1: Acquisition of candidate genes

[0078] Humicola fuscoatra NRRL 22980 (purchased from the U.S. Agricultural Research Service (NRRL)) was stored at room temperature on potato agar (PDA) medium (PDA medium composition: 200 g potatoes (peeled, diced, boiled for 10 min, filtrate collected), 20 g glucose, 15 g technical agar powder, diluted to 1 L with deionized water, and sterilized at 121°C for 30 min). A small amount of mycelium was inoculated into potato broth (PDB) medium (PDB medium composition: 200 g potatoes (peeled, diced, boiled for 10 min, filtrate collected), 20 g glucose, diluted to 1 L with deionized water, and sterilized at 121°C for 30 min) and cultured with shaking at 220 rpm at 28°C for 2 days. Mycelium was collected by filtration, ground in liquid nitrogen, and total DNA was extracted using the phenol-chloroform method. The extracted genome was sent to Shanghai Sangon Biotechnology Co., Ltd. for whole-genome sequencing using the Illumina HiSeq sequencing platform. 2500 system. Sequence analysis was performed using the software SOAPdenovo (version 2.04, http: / / soap.genomics.org.cn / soapdenovo.html). Finally, the gene cluster related to fuscoatroside biosynthesis was obtained using bioinformatics analysis (Figure 2A). FsoA (polypeptide sequence as shown in SEQ ID NO: 1, nucleic acid sequence as shown in SEQ ID NO: 6), FsoD (polypeptide sequence as shown in SEQ ID NO: 2, nucleic acid sequence as shown in SEQ ID NO: 7), FsoE (polypeptide sequence as shown in SEQ ID NO: 3, nucleic acid sequence as shown in SEQ ID NO: 8) and FsoF (polypeptide sequence as shown in SEQ ID NO: 4, nucleic acid sequence as shown in SEQ ID NO: 9) were selected as the final candidate research genes. In addition, an artificial fusion of EfuA was also designed. (TC) FsoA (GT) (The polypeptide sequence is shown in SEQ ID NO: 5, and the nucleic acid sequence is shown in SEQ ID NO: 10) ( FIG. 2B ).

[0079] Example 2: Construction of Aspergillus oryzae expression strain

[0080] Construction of Fuscoatroside Gene Expression Plasmid. fsoA, fsoD, fsoE and fsoF were amplified using the genomic DNA of strain Humicola fuscoatra NRRL 22980 as a template by the corresponding primer pairs Inf-fsoA-F (SEQ ID NO: 11) / Inf-fsoA-R (SEQ ID NO: 12), Inf-fsoD-F (SEQ ID NO: 13) / Inf-fsoD-R (SEQ ID NO: 14), Inf-fsoE-F (SEQ ID NO: 15) / Inf-fsoE-R (SEQ ID NO: 16), Inf-fsoF-F (SEQ ID NO: 17) / Inf-fsoF-R (SEQ ID NO: 18), and ligated to Aspergillus oryzae using the In fusion ligation method. oryzaeNSAR1, provided by Professor Ikuo Abe, University of Tokyo, Japan) expression plasmid pTAex3 or pUSA plasmid (provided by Professor Ikuo Abe, University of Tokyo, Japan) to form recombinant plasmids (pTAex3-fsoA, pTAex3-fsoD, pTAex3-fsoE, pTAex3-fsoF, pUSA-fsoD, pUSA-fsoE). Using the recombinant plasmids pTAex3-fsoD and pTAex3-fsoE as templates, the corresponding primer pairs Inf-pAdeA-Parm-F (SEQ ID NO: 19) / Inf-pTAex3-Tamy-R1 (SEQ ID NO: 20) and Inf-pTAex3-Parm-F1 (SEQ ID NO: 21) / Inf-pAdeA-Tamy-R (SEQ ID NO: 22) were used to amplify the DNA expression cassette containing the amylase amyB promoter (promoter) and terminator. Using the In Fusion ligation method, the cassette was ligated into the pAdeA plasmid (provided by Professor Ikuro Abe of the University of Tokyo, Japan) after digestion with XbaI to construct the two-gene expression plasmid pAdeA-fsoD-fsoE. Similarly, using the recombinant plasmids pTAex3-fsoD and pTAex3-fsoF as templates, the two-gene expression plasmid pAdeA-fsoD-fsoF was constructed in the same manner as described above.

[0081] The expression plasmid of artificial fusion enzyme was constructed. (TC) The gene was used as a template and the corresponding primers were used to amplify Inf-efuA (TC) -F(SEQ ID NO:23) / Inf-efuA (TC) -fsoA (GT)-R (SEQ ID NO: 24) amplified efuA (TC) fsoA (GT) The gene was used as a template and the corresponding primers were used to amplify Inf-efuA (TC) fsoA (GT) -F (SEQ ID NO: 25) / Inf-fsoA (GT) -R (SEQ ID NO: 26) amplified fsoA (GT) Next, the amplified efuA (TC) fsoA (GT) At the same time, it was connected to the expression plasmid pTAex3 to form the recombinant plasmid pTAex3-efuA (TC) fsoA (GT) .

[0082] Construction of FsoE mutant gene expression plasmid. Using the recombinant plasmid pTAex3-fsoE as a template, the corresponding mutant primer pairs FsoE-Y143A-F (SEQ ID NO: 27) / FsoE-Y143A-R (SEQ ID NO: 28), FsoE-F148A-F (SEQ ID NO: 29) / FsoE-F148A-R (SEQ ID NO: 30), FsoE-F259A-F (SEQ ID NO: 31) / FsoE-F259A-R (SEQ ID NO: 32), FsoE-R315A-F (SEQ ID NO: 33) / FsoE-R315A-R (SEQ ID NO: 34), FsoE-F337A-F (SEQ ID NO: 35) / FsoE-F337A-R (SEQ ID NO: 36), FsoE-W339A-F (SEQ ID NO: 37) / FsoE-W339A-R (SEQ ID NO: 38), FsoE-W339A-F (SEQ ID NO: 39) / FsoE-W339A-R (SEQ ID NO: 40), FsoE-W339A-F (SEQ ID NO: 41) / FsoE-W339A-R (SEQ ID NO: NO:37) / FsoE-W339A-R (SEQ ID NO:38), FsoE-N344A-F (SEQ ID NO:39) / FsoE-N344A-R (SEQ ID NO:40), FsoE-C517F-F (SEQ ID NO:41) / FsoE-C517A-R (SEQ ID NO:42), FsoE-F557A-F (SEQ ID NO:43) / FsoE-F557A-R (SEQ ID NO:44), FsoE-Y143F-F (SEQ ID NO:45) / FsoE-Y143A-R (SEQ ID NO:28) amplification, using In The fusion connection method was used to connect to the Aspergillus oryzae expression plasmid pTAex3 to form mutant recombinant plasmids (pTAex3-fsoE-Y143A, pTAex3-fsoE-F148A, pTAex3-fsoE-F259A, pTAex3-fsoE-R315A, pTAex3-fsoE-F337A, pTAex3-fsoE-W339A, pTAex3-fsoE-N344A, pTAex3-fsoE-C517A, pTAex3-fsoE-F557A, pTAex3-fsoE-Y143F).

[0083] Example 3: Aspergillus oryzae (A. oryzae NSAR1) protoplast preparation and transfection.

[0084] 1) 20 μL of Aspergillus oryzae spore preservation solution was added to 10 mL of DPY medium (DPY medium composition: 20 g dextrin, 10 g polypeptone, 5 g yeast extract, 0.5 g magnesium sulfate heptahydrate, 5 g potassium dihydrogen phosphate, 0.1 g adenine, dilute to 1 L with deionized water, and sterilize at 121°C for 30 minutes). The culture was shaken at 28°C, 200 rpm for 1-2 days.

[0085] 2) Add the above bacterial culture solution to 100 mL of DPY medium and culture at 28°C, 200 rpm, and shake for 1-2 days.

[0086] 3) Take approximately 15 mL of the bacterial solution and filter it through a sterilized syringe filter. Press the cells dry. Remove the cells with a sterilized spatula and place them into a new 50 mL centrifuge tube. Add 10 mL of TF solution 1 (TF solution 1 composition: maleic acid 0.058 g, ammonium sulfate 0.79 g, yatalase 0.1 g, pH 5.5) that has been sterilized by filtration through a 0.22 μm microporous membrane.

[0087] 4) Shake in a 30°C incubator for 3 hours until the supernatant is visibly turbid and pale red. Filter the protoplasts into a 50 mL test tube using a syringe filter. If clogging occurs, poke the cotton surface with a bamboo stick.

[0088] 5) Add an equal amount of TF solution 2 (TF solution 2 composition: sorbitol 87.4 g, calcium chloride dihydrate 2.94 g, sodium chloride 0.82 g, 1 M Tris-HCl (pH 7.5) 4 mL, dilute to 400 mL with deionized water, and autoclave at 121°C for 30 min) (10 mL), gently mix by inverting the tube, and centrifuge at 1500 rpm at 4°C for 10 min.

[0089] 6) Remove the supernatant, add 5 mL of TF solution 2, centrifuge at 4°C, 1500 rpm for 10 minutes, remove the supernatant, and add an appropriate amount of TF solution 2 to make the protoplast concentration 1-5×10 7 / mL, invert upside down to suspend.

[0090] 7) Take 200 μL of the protoplast solution into a 15 mL centrifuge tube, add 10 μL of the corresponding expression plasmid at a concentration of 1 μg / μL, and mix gently.

[0091] 8) Let stand on ice for 30 minutes. During this time, dissolve the M selection medium (upper and lower layers) in a microwave oven and keep warm in a 50°C water bath.

[0092] 9) Add 250 μL, 250 μL, and 850 μL of TF solution 3 (TF solution 3 composition: PEG 4000 120 g, calcium chloride dihydrate 1.47 g, 1 M Tris-HCl (pH 7.5) 2 mL, dilute to 200 mL with deionized water, and sterilize at 121°C for 30 min) to the suspension in 7 in three separate additions. Mix thoroughly by pipetting after each addition and let stand at room temperature for 20 min.

[0093] 10) Add 5 mL of TF solution 2 and gently mix by inverting the tube.

[0094] 11) Centrifuge at 1500 rpm for 10 min at 4°C. Remove the supernatant and add 200 μL of TF solution 2. Gently mix with a 1 mL pipette and add to the center of a culture dish containing lower M screening medium (lower M screening medium composition: 0.5 g potassium chloride, 0.5 g sodium chloride, 2 g ammonium chloride, 1 g ammonium sulfate, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.02 g ferrous sulfate heptahydrate, 20 g glucose, 15 g technical agar powder, 218.6 g sorbitol, plus the appropriate screening nutrients (e.g., when introducing a single plasmid: pTAex3 plasmid → 1.5 g methionine + 0.1 g adenine; pUSA plasmid → 1 g arginine + 0.1 g adenine; pAdeA plasmid → 1.5 g methionine + 1 g arginine), dilute to 1 L with deionized water, pH 5.5, and sterilize at 121°C for 30 min). Quickly add 5 mL of 50°C insulated upper M screening medium around the culture dish (composition of upper M screening medium: 0.5 g potassium chloride, 0.5 g sodium chloride, 2 g ammonium chloride, 1 g ammonium sulfate, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.02 g ferrous sulfate heptahydrate, 20 g glucose, 8 g technical agar powder, 218.6 g sorbitol, and the corresponding screening nutrients (e.g., when introducing a single plasmid: pTAex3 plasmid → 1.5 g methionine + 0.1 g adenine; pUSA plasmid → 1 g arginine + 0.1 g adenine; pAdeA plasmid → 1.5 g methionine + 1 g arginine), dilute to 1 L with deionized water, and sterilize at 121°C for 30 minutes) and quickly mix.

[0095] 12) After the plate is air-dried, seal it with parafilm, place it upside down in an incubator, and culture it at 28°C for 3-5 days. Subsequently, select the transformed Aspergillus oryzae strain and inoculate it into M stable medium (M stable medium composition: 0.5 g potassium chloride, 0.5 g sodium chloride, 2 g ammonium chloride, 1 g ammonium sulfate, 1 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.02 g ferrous sulfate heptahydrate, 15 g technical agar powder, 20 g glucose, add the corresponding selection nutrients (e.g., for a single plasmid: pTAex3 plasmid → 1.5 g methionine + 0.1 g adenine; pUSA plasmid → 1 g arginine + 0.1 g adenine; pAdeA plasmid → 1.5 g methionine + 1 g arginine), dilute to 1 L with deionized water, and sterilize at 121°C for 30 minutes) for passage 1-2 times.

[0096] Example 4: Metabolite Analysis of the Aspergillus oryzae Expression Strain Used for Fuscoatroside Synthesis

[0097] The Aspergillus oryzae (prepared according to Example 3) transformant expressing the target gene was inoculated into 10mL DPY culture medium, and 28°C, 200rpm shaking culture was used for 2 days as seed liquid. The seed liquid was then inoculated into 100mL CD-starch culture medium (CD-starch culture medium composition: sodium nitrate 3g, potassium chloride 2g, magnesium sulfate heptahydrate 0.5g, potassium dihydrogen phosphate 1g, ferrous sulfate heptahydrate 0.02g, polypeptone 10g, soluble starch 20g, adenine 0.1g, pH 5.5, deionized water was settled to 1L, and sterilized at 121°C for 30 minutes), 28°C, 200rpm shaking culture for 5 days. After fermentation was complete, mycelium was collected using a funnel filtration, and the mycelium was pressed dry, then anhydrous ethanol was added to soak overnight. The mycelium after soaking was ultrasonically extracted for 30min, concentrated under reduced pressure, and the extract was dissolved in methanol for LC-MS analysis. The results are shown in Figures 3 and 4. FsoA can catalyze the production of fernane-type skeleton compound 2 and C3 glucose fernane-type skeleton compound 3 in Aspergillus oryzae. It is determined that the TC domain in the fusion FsoA is responsible for the formation of the fernane-type skeleton with a double bond at the C8-C9 position, while the GT domain is responsible for the β-D-pyranose glucose glycosylation on the α-hydroxyl at the C3 position. FsoA and FsoD work together in Aspergillus oryzae to produce compounds 4 and 5. It is determined that FsoD is responsible for the oxidation of C2 to form an α-hydroxyl. FsoA and FsoE in Aspergillus oryzae The combined action of FsoA, FsoD and FsoE in Aspergillus oryzae produced compounds 6 and 7, confirming that FsoE is responsible for catalyzing the cleavage of the C19-C20 carbon bond, forming a structure with a carboxyl group on the left and a methyl group on the right; FsoA, FsoD and FsoE in Aspergillus oryzae combined to produce compounds 8, 9, 10 and 11; FsoA, FsoD and FsoF in Aspergillus oryzae combined to produce compound 12; the four genes FsoA, FsoD, FsoE and FsoF are fully sufficient to synthesize fuscoatroside in Aspergillus oryzae (1).

[0098] LC-MS conditions were as follows:

[0099] LC-MS analysis was performed using a Dionex UltiMate 3000 equipped with an UltiMate 3000 Diode Array Detector and a Bruker amaZon SL APCI source low-resolution mass spectrometer. The analytical column was a COSMOSIL 5μm C18 column (5μm, 4.6×150mm). The mobile phase consisted of deionized water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid). The gradient was 50% to 100% acetonitrile (0-10 min) and 100% to 100% acetonitrile (10-50 min); the flow rate was 1 mL / min.

[0100] Example 5: Metabolite analysis of the Aspergillus oryzae expression strain used to synthesize the anemaphene gold precursor (13).

[0101] The Aspergillus oryzae transformed strain expressing the target gene (prepared according to Example 3) was inoculated into 10 mL DPY medium and cultured at 28 ° C., 200 rpm shaking for 2 days as a seed solution. The seed solution was then inoculated into 100 mL CD-starch medium and cultured at 28 ° C., 200 rpm shaking for 6 days. After fermentation was complete, the mycelium was soaked overnight in anhydrous ethanol, ultrasonically extracted for 30 min, and the culture medium was extracted with ethyl acetate. The extracts of the mycelium and the culture medium were mixed together, concentrated under reduced pressure, and finally dissolved in chromatographic methanol for ELSD analysis. As shown in Figure 5, efuA was simultaneously expressed in Aspergillus oryzae. (TC) fsoA (GT) , fsoD, fsoE and fsoF can produce anthracene precursors (13), and the artificial fusion enzyme EfuA was identified. (TC) FsoA (GT) It is responsible for the formation of a double-bonded fernane-type skeleton at C9-C11 with β-D-pyranose linked to C3.

[0102] The ELSD conditions are as follows:

[0103] ELSD analysis was performed using a Dionex UltiMate 3000 equipped with an UltiMate 3000 Diode Array Detector and an Alltech (Grace) 2000ES ELSD detector. The liquid chromatography column was a COSMOSIL 5μm C18 column (5μm, 4.6×150mm). The mobile phase consisted of deionized water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid). The gradient was 50% to 100% acetonitrile (0-30 min) and 100% to 100% acetonitrile (30-50 min); the flow rate was 1 mL / min.

[0104] Example 6: Substrate feeding experiment of P450 oxidase FsoE

[0105] According to the method in embodiment 3, the aspergillus oryzae transfection strain only containing pTAex3-fsoE is constructed.First, 10mL DPY culture medium is inoculated containing aspergillus oryzae single gene strain (control group is blank aspergillus oryzae), 28 DEG C, 200rpm are cultivated 2-3 days.Then, above-mentioned culture fluid is joined in 100mL CD-starch culture medium and induces target gene expression, 28 DEG C, after cultivating 1 day at 200rpm, in culture medium, add the substrate (compound 9 or compound 10) that DMSO dissolves, then continue to cultivate 4 days.Finally, mycelium is collected with funnel filtration, and thalline is pressed dry, then dehydrated alcohol is added to soak thalline, ultrasonic extraction, concentrating under reduced pressure, extract is analyzed for HPLC after chromatogram methanol dissolution.As a result as shown in Figure 6, FsoE can catalyze compound 9 to produce compound 10 and 11 in aspergillus oryzae, and can catalyze compound 10 to produce compound 11 again. The above results indicate that the FsoE-mediated E-ring cleavage process first oxidizes C19 to C19β-hydroxyl, then to C19 carbonyl, and finally to C19-C20 cleavage.

[0106] HPLC conditions are as follows:

[0107] HPLC analysis was performed using a Dionex UltiMate 3000 equipped with an UltiMate 3000 Diode Array Detector. The analytical column was a COSMOSIL 5μm C18 column (5μm, 4.6×150mm). The mobile phase consisted of deionized water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid). The gradient was 50% to 100% acetonitrile (0-30 min) and 100% to 100% acetonitrile (30-50 min); the flow rate was 1 mL / min.

[0108] Example 7: Point mutation experiment of P450 oxidase FsoE

[0109] We used AlphaFold2 and AlphaFill to construct an enzyme model of FsoE, in which heme is covalently linked to the conserved residue C517. Then, using AutoDock to perform molecular docking on substrate 10, we found a hydrophobic active pocket surrounded by 8 different amino acid residues (R315, F148, F259, F337, W339, F557, N344, Y143) (Figure 7A). Based on the molecular docking results, we performed point mutation experiments on these residues and studied the catalytic function of key residues through feeding experiments. According to the method in Example 3, an Aspergillus oryzae transfected strain containing only the pTAex3-fsoE mutant plasmid was constructed. First, the Aspergillus oryzae single gene strain (the control group was blank Aspergillus oryzae) was inoculated into 10mL DPY medium and cultured at 28°C and 200rpm for 2-3 days. The culture medium was then added to 100 mL of CD-starch medium to induce target gene expression. After incubation at 28°C and 200 rpm for one day, compound 9 dissolved in DMSO was added to the medium, and incubation continued for another 3-4 days. Finally, the mycelia were collected by filtration using a funnel, squeezed dry, and then soaked in anhydrous ethanol. Ultrasonic extraction was performed and the extract was concentrated under reduced pressure. The extract was dissolved in methanol and analyzed by LC-MS. As shown in Figure 7B, mutations of N344 and R315 to A completely abolished the enzymatic activity of FsoE, suggesting that R315 and N344 may play a key role by forming hydrogen bonds with the C3 hydroxyl group and C19 carbonyl group of the substrate. Furthermore, mutations of two non-polar residues, W339 and F337, also completely abolished the catalytic activity of FsoE, suggesting that these active sites may bind the substrate through hydrophobic interactions. Furthermore, mutations of four residues near the E-loop (F557A, F259A, F148A, and Y143A) still produced the C19 carbonyl product 10, and F557A almost converted substrate 9 to compound 10. However, all four mutants failed to produce E-loop cleavage products, indicating that F557, F259, F148, and Y143 play a key role in catalyzing C-C bond cleavage to form the ring-opening product 11. Furthermore, when Y at position 143 was mutated to F, the results showed that the Y143F mutant could only convert substrate 9 to compound 10 but not compound 11, indicating that the hydroxyl group on Y143 is important for C-C bond cleavage.

[0110] LC-MS analysis was performed using a Dionex UltiMate 3000 equipped with an UltiMate 3000 Diode Array Detector and a Bruker amaZon SL APCI source low-resolution mass spectrometer. The analytical column was a COSMOSIL 5μm C18 column (5μm, 4.6×150mm). The mobile phase consisted of deionized water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid). The gradient was 50% to 100% acetonitrile (0-30 min) and 100% to 100% acetonitrile (30-50 min); the flow rate was 1 mL / min.

[0111] Example 8: Isolation, purification and structural identification of compounds

[0112] Example 8.1 Isolation and purification of compounds

[0113] The methods and results of compound analysis and purification involved in the above examples are summarized as follows:

[0114] Isolation and purification of compound 1: 5 L of the transfected strain containing fsoADEF was fermented. The mycelium was soaked in anhydrous ethanol overnight and ultrasonically extracted for 30 minutes, repeated three times. The culture medium was extracted twice with ethyl acetate, repeated three times. The mycelial and culture medium extracts were combined and eluted by ODS column chromatography (methanol-water → 30%, 50%, 70%, 90%, and 100% v / v) to yield five fractions. Fraction 4 was purified by HPLC (60% acetonitrile-water, 0.1% formic acid, 3 mL / min) to yield the title compound 1.

[0115] Isolation and purification of compound 2: 1 L of the transfected strain harboring fsoA was fermented, the cells harvested, and soaked in anhydrous ethanol overnight. Ultrasonic extraction was repeated three times for 30 minutes. The extract was eluted through a silica gel column (cyclohexane-ethyl acetate → 100:0, 98:2, 95:5, 90:10, 80:20, ethyl acetate: methanol v / v) to yield seven fractions. Target compound 2 was obtained from fraction 3.

[0116] Isolation and purification of compound 3: Weigh aglycone compound 2 (20 mg) and D-glucose trichloroacetimidate glycosyl donor (50 mg) and add them together into a 50 mL dried round-bottom flask, then add an appropriate amount of Molecular sieves were added, followed by 10 mL of dry dichloromethane. The mixture was stirred on ice for 20 minutes. When the system temperature dropped to 0°C, 50 μL of the catalyst TMSOTf was added dropwise. The reaction was continued on ice for 2-3 hours, then at room temperature overnight. The reaction progress was monitored by TLC. After completion, a few drops of triethylamine were added to terminate the reaction. The reaction was then filtered and concentrated under reduced pressure to obtain a crude sample. The crude sample obtained above was dissolved in 10 mL of a 1:1 methanol / dichloromethane mixture, and 80 mg of sodium methoxide was added to adjust the pH of the reaction system to >9. The reaction was allowed to proceed overnight at room temperature. The reaction progress was monitored by TLC. After completion, an acidic cation exchange resin was added to neutralize the reaction system to a neutral pH. The sample was then filtered and concentrated under reduced pressure to obtain a crude sample with the deprotected group removed. Finally, the glycosyl compound 3 was separated by silica gel column chromatography (the target compound was in the ethyl acetate layer) and prepared by HPLC (100% methanol, 3 mL / min). Finally, LC-MS comparison confirmed that compound 3 obtained by the above glycosylation was metabolite 3 in the Aspergillus oryzae fsoA transfected strain.

[0117] Isolation and purification of compound 4: Compound 12 was dissolved in 12 mL of a mixed solvent of methanol / dichloromethane (1:1), and 80 mg of sodium methoxide was added to a pH value of >9. The reaction was stirred at room temperature and allowed to react overnight. TLC was used to monitor the progress of the reaction. After the reaction was completed, an acidic cation exchange resin was added for neutralization to neutralize the pH value of the system. The mixture was then filtered and concentrated under reduced pressure. HPLC preparation (100% methanol, 3 mL / min) was used to obtain mixture 4. Compound 4 was then obtained by secondary preparation and purification using HPLC (90% acetonitrile: 10% tetrahydrofuran, 3 mL / min). Finally, LC-MS comparison confirmed that compound 4 obtained by the above deacetylation was metabolite 4 in the Aspergillus oryzae fsoAD transfectant strain.

[0118] Isolation and purification of compound 5: 5 L of the transfected strain containing fsoAD was fermented, the cells harvested, and soaked in anhydrous ethanol overnight. Ultrasonic extraction was repeated three times for 30 min. The extract was eluted through a silica gel column (cyclohexane-ethyl acetate → 100:0, 95:5, 90:10, 80:20, 70:30, 50:50, ethyl acetate: methanol v / v) to yield eight fractions. Target compound 5 was obtained from fraction 5.

[0119] Isolation and purification of compounds 6 and 7: 5 L of the transfected strain containing fsoAE was fermented, the cells harvested, soaked in anhydrous ethanol overnight, and ultrasonically extracted for 30 min, repeated three times. The extract was eluted through a silica gel column (cyclohexane-ethyl acetate → 100:0, 95:5, 90:10, 80:20, 70:30, 60:40, 50:50, ethyl acetate, methanol v / v) to yield nine fractions. Fraction 8 was purified by HPLC (85% acetonitrile-water, 0.1% formic acid, 3 mL / min) to yield target compound 6. Fraction 4 was purified by HPLC (100% methanol, 3 mL / min) to yield target compound 7.

[0120] Isolation and purification of compound 8: Compound fuscoatroside (1) was dissolved in 12 mL of a mixed solvent of methanol / dichloromethane (1:1), and 80 mg of sodium methoxide was added to make the pH value of the reaction system > 9. The mixture was stirred at room temperature and allowed to react overnight. The reaction progress was monitored by TLC. After the reaction was completed, an acidic cation exchange resin was added for neutralization to make the pH value of the system neutral. The mixture was then filtered and concentrated under reduced pressure. Compound 8 was obtained by HPLC preparation (60% acetonitrile-water, 0.1% formic acid, 3 mL / min). Finally, LC-MS comparison confirmed that compound 8 obtained by the above deacetylation was metabolite 8 in the Aspergillus oryzae fsoADE transfected strain.

[0121] Isolation and purification of compounds 9, 10, and 11: 10 L of the transfected strain containing fsoADE was fermented, the cells harvested, soaked in anhydrous ethanol overnight, and ultrasonically extracted for 30 min, repeated three times. The extract was eluted through a silica gel column (cyclohexane-ethyl acetate → 100:0, 90:10, 85:15, 80:20, 70:30, 50:50, ethyl acetate: methanol v / v) to yield eight fractions. Fraction 6 was purified by HPLC (85% acetonitrile-water, 0.1% formic acid, 3 mL / min) to yield target compounds 9 and 11. Fraction 3 was purified by HPLC (88% acetonitrile-water, 0.1% formic acid, 3 mL / min) to yield target compound 10.

[0122] Isolation and purification of compound 12: 5 L of the transfected strain containing fsoADF was fermented, the cells harvested, and soaked in anhydrous ethanol overnight. Ultrasonic extraction was repeated three times for 30 min. The extract was eluted through a silica gel column (cyclohexane-ethyl acetate → 90:10, 85:15, 80:20, 75:25, 65:35, 50:50, ethyl acetate: methanol v / v) to yield eight fractions. Fraction 8 was purified by HPLC (100% methanol, 3 mL / min) to yield the target compound 12.

[0123] Isolation and purification of compound 13: Fermentation 5L contains efuA (TC) fsoA(GT) The transfected strain was cultured at 28°C, 220 rpm, for 6 days, then filtered to separate the cells from the culture medium. The culture medium was extracted three times with ethyl acetate and eluted via ODS column chromatography (methanol-water, 0.1% formic acid → 30%, 50%, 70%, 85%, 90%, 95%, and 100% v / v) to yield seven fractions. Fraction 5 was purified by HPLC (70% acetonitrile-water, 0.1% formic acid, 3 mL / min) to yield the title compound 13.

[0124] Example 8.2 Structure, name, number and NMR confirmation data of the compounds involved in the examples

[0125] Assignment of the NMR data of compound 1 (solvent: deuterated pyridine, 150 MHz carbon spectrum, 600 MHz hydrogen spectrum)

[0126] a Unresolvable signals due to overlapping or complex multiplicities were reported without specifying the multiplicity.

[0127] Assignment of the NMR data of compound 2 (solvent: deuterated chloroform, 100 MHz carbon spectrum, 400 MHz hydrogen spectrum)

[0128] a Unresolvable signals due to overlapping or complex multiplicities were reported without specifying the multiplicity.

[0129] Assignment of the NMR data of compound 3 (solvent: deuterated pyridine, 150 MHz carbon spectrum, 600 MHz hydrogen spectrum)

[0130] a Unresolvable signals due to overlapping or complex multiplicities were reported without specifying the multiplicity.

[0131] Assignment of the NMR data of compound 4 (solvent: deuterated pyridine, 150 MHz carbon spectrum, 600 MHz hydrogen spectrum)

[0132] a Unresolvable signals due to overlapping or complex multiplicities were reported without specifying the multiplicity.

[0133] Assignment of the NMR data of compound 5 (solvent: deuterated chloroform, 100 MHz carbon spectrum, 400 MHz hydrogen spectrum)

[0134] a Unresolvable signals due to overlapping or complex multiplicities were reported without specifying the multiplicity.

[0135] b These data were collected on a 600 MHz NMR spectrometer.

[0136] Assignment of the NMR data of compound 6 (solvent: deuterated pyridine, 150 MHz carbon spectrum, 600 MHz hydrogen spectrum)

[0137] a Unresolvable signals due to overlapping or complex multiplicities were reported without specifying the multiplicity.

[0138] Assignment of the NMR data of compound 7 (solvent: deuterated chloroform, 150 MHz carbon spectrum, 600 MHz hydrogen spectrum)

[0139] a Unresolvable signals due to overlapping or complex multiplicities were reported without specifying the multiplicity.

[0140] Assignment of the NMR data of compound 8 (solvent: deuterated pyridine, 150 MHz carbon spectrum, 600 MHz hydrogen spectrum)

[0141] a Unresolvable signals due to overlapping or complex multiplicities were reported without specifying the multiplicity.

[0142] Assignment of the NMR data of compound 9 (solvent: deuterated chloroform, 150 MHz carbon spectrum, 600 MHz hydrogen spectrum)

[0143] a Unresolvable signals due to overlapping or complex multiplicities were reported without specifying the multiplicity.

[0144] Assignment of the NMR data of compound 10 (solvent: deuterated chloroform, 150 MHz carbon spectrum, 600 MHz hydrogen spectrum)

[0145] a Unresolvable signals due to overlapping or complex multiplicities were reported without specifying the multiplicity.

[0146] Assignment of the NMR data of compound 11 (solvent: deuterated chloroform, 150 MHz carbon spectrum, 600 MHz hydrogen spectrum)

[0147] a Unresolvable signals due to overlapping or complex multiplicities were reported without specifying the multiplicity.

[0148] Assignment of the NMR data of compound 12 (solvent: deuterated pyridine, 150 MHz carbon spectrum, 600 MHz hydrogen spectrum)

[0149] a Unresolvable signals due to overlapping or complex multiplicities were reported without specifying the multiplicity.

[0150] Assignment of the NMR data of compound 13 (solvent: deuterated pyridine, 150 MHz carbon spectrum, 600 MHz hydrogen spectrum)

[0151] a Unresolvable signals due to overlapping or complex multiplicities were reported without specifying the multiplicity.

[0152] The sequence involved in the present invention is as follows:

[0153] Amino acid sequence

[0154] >FsoA (SEQ ID NO: 1)

[0155] >FsoD (SEQ ID NO: 2)

[0156] >FsoE (SEQ ID NO: 3)

[0157] >FsoF (SEQ ID NO: 4)

[0158] >EfuA (TC) FsoA (GT) (SEQ ID NO:5)

[0159] Nucleotide sequence (DNA)

[0160] >fsoA (SEQ ID NO: 6)

[0161] >fsoD (SEQ ID NO: 7)

[0162] >fsoE (SEQ ID NO: 8)

[0163] >fsoF (SEQ ID NO: 9)

[0164] >efuA (TC) fsoA (GT) (SEQ ID NO: 10)

[0165] Primer sequences

[0166] >Inf-fsoA-F (SEQ ID NO: 11)

[0167] >Inf-fsoA-R (SEQ ID NO: 12)

[0168] >Inf-fsoD-F (SEQ ID NO: 13)

[0169] >Inf-fsoD-R (SEQ ID NO: 14)

[0170] >Inf-fsoE-F (SEQ ID NO: 15)

[0171] >Inf-fsoE-R (SEQ ID NO: 16)

[0172] >Inf-fsoF-F (SEQ ID NO: 17)

[0173] >Inf-fsoF-R (SEQ ID NO: 18)

[0174] >Inf-pAdeA-Parm-F(SEQ ID NO:19)

[0175] >Inf-pTAex3-Tamy-R1(SEQ ID NO:20)

[0176] >Inf-pTAex3-Parm-F1(SEQ ID NO:21)

[0177] >Inf-pAdeA-Tamy-R(SEQ ID NO:22)

[0178] >Inf-efuA (TC) -F(SEQ ID NO:23)

[0179] >Inf-efuA (TC) fsoA (GT) -R(SEQ ID NO:24)

[0180] >Inf-efuA (TC) fsoA (GT) -F(SEQ ID NO:25)

[0181] >Inf-fsoA (GT) -R(SEQ ID NO:26)

[0182] >FsoE-Y143A-F(SEQ ID NO:27)

[0183] >FsoE-Y143A-R(SEQ ID NO:28)

[0184] >FsoE-F148A-F(SEQ ID NO:29)

[0185] >FsoE-F148A-R(SEQ ID NO:30)

[0186] >FsoE-F259A-F(SEQ ID NO:31)

[0187] >FsoE-F259A-R(SEQ ID NO:32)

[0188] >FsoE-R315A-F(SEQ ID NO:33)

[0189] >FsoE-R315A-R(SEQ ID NO:34)

[0190] >FsoE-F337A-F(SEQ ID NO:35)

[0191] >FsoE-F337A-R(SEQ ID NO:36)

[0192] >FsoE-W339A-R(SEQ ID NO:38)

[0193] >FsoE-N344A-F(SEQ ID NO:39)

[0194] >FsoE-N344A-R(SEQ ID NO:40)

[0195] >FsoE-C517A-F(SEQ ID NO:41)

[0196] >FsoE-C517A-R(SEQ ID NO:42)

[0197] >FsoE-F557A-F(SEQ ID NO:43)

[0198] >FsoE-F557A-R(SEQ ID NO:44)

[0199] >FsoE-Y143F-F(SEQ ID NO:45)

[0200] References:

[0201] 1.Schwartz,R.E.;Smith,S.K.;Onishi,J.C.;Meinz,M.;Kurtz,M.;Giacobbe,R.A.;Wilson,K.E.;Liesch,J.;Zink,D.;Horn,W.;Morris,S.;Cabello,A.;Vicente,F.Isolation and Structural Determination of Enfumafungin,a Triterpene Glycoside Antifungal Agent That is a Specific Inhibitor of Glucan Synthesis.J.Am.Chem.Soc.2000,122,4882-4886.

[0202] 2.Joshi,B.K.;Gloer,J.B.;Wicklow,D.T.Bioactive Natural Products from a Sclerotium-Colonizing Isolate of Humicola fuscoatra.J.Nat.Prod.2002,65,1734-1737.

[0203] 3.Deyrup,S.T.;Gloer,J.B.;O’Donnell,K.;Wicklow,D.T.Kolokosides A-D:Triterpenoid Glycosides from a Hawaiian Isolate of Xylaria sp.J.Nat.Prod.2007,70,378-382.

[0204] 4.Shigematsu,N.;Tsujii,E.;Kayakiri,N.;Takase,S.;Tanaka,H.;Tada,T.WF11605,an Antagonist of Leukotriene B4 Produced by a Fungus.J.Antibiot.(Tokyo)1992,45,704-708.

[0205] 5.Phillips,N.A.;Rocktashel,M.;Merjanian,L.Ibrexafungerp for the Treatment of Vulvovaginal Candidiasis:Design,Development and Place in Therapy.Drug Des.Devel.Ther.2023,17,363-367.

[0206] 6.Kuhnert,E.;Li,Y.;Lan,N.;Yue,Q.;Chen,L.;Cox,R.J.;An,Z.;Yokoyama,K.;Bills,G.F.Enfumafungin Synthase Represents a Novel Lineage of Fungal Triterpene Cyclases.Environ.Microbiol.2018,20,3325-3342.

Claims

1. An isolated or synthetic polypeptide, characterized in that The polypeptide is selected from: a) a FsoA polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 90% sequence identity thereto, b) a FsoD polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least 90% sequence identity thereto, c) a FsoE polypeptide comprising the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having at least 90% sequence identity thereto, d) a FsoF polypeptide comprising the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 90% sequence identity thereto, and e) EfuA comprising the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having at least 90% sequence identity thereto (TC) FsoA (GT) polypeptide.

2. A polynucleotide, characterized in that The polynucleotide encodes the polypeptide of claim 1.

3. The polynucleotide according to claim 2, characterized in that The polynucleotide is selected from the group consisting of: (i) a nucleotide sequence comprising the sequence shown in SEQ ID NO: 6 or a sequence having at least 90% sequence identity thereto; (ii) a nucleotide sequence comprising the sequence shown in SEQ ID NO: 7 or a sequence having at least 90% sequence identity thereto; (iii) a nucleotide sequence comprising the sequence shown in SEQ ID NO: 8 or a sequence having at least 90% sequence identity thereto; (iv) a nucleotide sequence comprising the sequence shown in SEQ ID NO: 9 or a sequence having at least 90% sequence identity thereto; and (v) a nucleotide sequence comprising the sequence shown in SEQ ID NO: 10 or a sequence having at least 90% sequence identity thereto.

4. A carrier, characterized in that The vector comprises the polynucleotide according to claim 2 or 3.

5. A host cell, characterized in that The cell comprises the vector according to claim 4; preferably, the cell is a fungal cell or a bacterial cell; more preferably, the fungal cell is selected from Humicola fuscoatra and Aspergillus oryzae cells; the bacterial cell is an Escherichia coli cell.

6. A method for the biosynthesis of fuscoatroside (1) or a precursor thereof, comprising contacting one or more of the polypeptides of a) to d) of claim 1 with 2,3(S)-epoxysqualene and uridine diphosphate glucose (UDPG) substrate; preferably, the synthesis method comprises: Expressing one or more of the polypeptides a) to d) of claim 1 in a host cell, preferably an Aspergillus oryzae host cell, so that it catalyzes the synthesis of fuscoatroside or its precursor from a substrate; and isolating fuscoatroside or its precursor from the host cell, wherein the structure of the compound fuscoatroside is as follows:

7. A biosynthetic method of an anergine precursor compound 13 or a precursor thereof, comprising contacting one or more of the polypeptides of b) to e) of claim 1 with 2,3(S)-epoxysqualene and uridine diphosphate glucose (UDPG) substrate; preferably, the synthesis method comprises: Expressing one or more of the polypeptides of b) to e) of claim 1 in a host cell, preferably an Aspergillus oryzae host cell, so that it catalyzes the synthesis of compound 13 or its precursor from a substrate; and isolating compound 13 or its precursor from the host cell, wherein the structure of compound 13 is as follows:

8. A polypeptide that catalyzes cleavage of the E-ring C19-C20 position of a fernane-type compound, comprising amino acid residues corresponding to R315, F148, F259, F337, W339, F557, N344, and Y143 of the amino acid sequence shown in SEQ ID NO:

3.

9. A method for catalyzing cleavage of the E-ring C19-C20 of a fernane-type compound, comprising contacting the FsoE polypeptide of claim 1 or the polypeptide of claim 10 with the fernane-type compound; preferably, the method comprises expressing the FsoE polypeptide of claim 1 or the polypeptide of claim 10 in a host cell, preferably an Aspergillus oryzae host cell, so that the polypeptide catalyzes cleavage of the E-ring C19-C20 of the fernane-type compound.

10. The method according to claim 9, wherein the structure of the fernane-type compound is:

11. A catalytic site of a polypeptide that catalyzes the cleavage of the E-ring C19-C20 position of a fernane-type compound, which is the amino acid residues corresponding to R315, F148, F259, F337, W339, F557, N344 and Y143 of the amino acid sequence shown in SEQ ID NO:

3.

12. Use of the polypeptide according to claim 1 or 8, the polynucleotide according to claim 2 or 3, the vector according to claim 4 or the host cell according to claim 5 in the synthesis of anafenacin antibiotics.

13. A kit, characterized in that Comprising the polypeptide of claim 1 or 8, the polynucleotide of claim 2 or 3, the vector of claim 4, or the host cell of claim 5.

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