Diketopiperazine heterodimer as well as biosynthesis method and application thereof

Abstract

The invention discloses a diketopiperazine heterodimer as well as a biosynthesis method and application thereof, and belongs to the technical field of bioengineering. The biosynthesis method of the diketopiperazine heterodimer comprises the following steps: S1, culturing a recombinant cell containing P450 dimerization enzyme or a mutant thereof, and performing induced expression to obtain a whole-cell culture; and S2, adding a cyclic dipeptide substrate containing tryptophan into the whole-cell culture, carrying out a catalytic reaction, and separating and purifying the obtained product to obtain the diketopiperazine heterodimer. According to the invention, the P450 dimerization enzyme or the mutant thereof is used for catalyzing the biosynthesis of the tryptophan-containing diketopiperazine into the diketopiperazine heterodimer with different regioselectivity / stereoselectivity, the biosynthesis method is green and environment-friendly, the efficiency is high, and the variety of the diketopiperazine heterodimer is greatly expanded.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a diketopiperazine heterodimer, its biosynthesis method, and its application. Background Technology

[0002] Natural products derived from tryptophan-containing diketopiperazine (TDKP) are a large class of secondary metabolites with broad biological activities produced by plants, mammals, fungi, and bacteria. [1–3] Among them, diketopiperazine dimers containing tryptophan (DTDKPs) exhibit higher pharmacophore concentrations when binding to receptors associated with several human diseases due to their unique dimer structure, or due to increased affinity for receptors through dual interactions between dimers. Therefore, compared to their corresponding monomeric structures, they possess better biopharmaceutical activities, such as anti-inflammatory, anticancer, antimalarial, antiviral, and neuroprotective effects. [1–8] The vast majority of naturally derived DTDKPs are composed of C3. sp3 -C3´ sp3 Two TDKP monomers are linked by a bond to form two homodimers with consecutive quaternary stereocenters of the same configuration. Only in a few special cases are the monomer units linked by unusual bonds, such as C3. sp3 -C6´ / C7´ / C8´ sp2 C3 sp3 -N1´ or N1-C7´ sp2 Connecting to form structurally diverse asymmetric dimers [1–5,8,9] This greatly limits the structural diversity of DTDKPs, making it difficult to provide more options for new drug screening.

[0003] Chemical synthesis methods often involve multi-step reactions and require heavy metal catalysts (such as (CuOTf)2·PhMe, CoCl(PPh3)3, or AgSbF6) or harsh conditions such as high temperature or vacuum, resulting in low product yields, high catalyst costs, and environmental pollution. [9–11] The inherently low selectivity of chemical methods makes the chemical synthesis of complex asymmetric dimers a significant challenge, with overall yields remaining at only around 10%. [12–14] Therefore, the search for novel, efficient, and green synthetic DTDKPs with diverse structures has significant scientific and economic value.

[0004] Compared with chemical catalysis, biosynthetic methods using enzymes or organisms as catalysts have advantages such as mild reaction conditions, high selectivity, high efficiency, and being environmentally friendly. This has led to a greater focus in recent years on exploring the biocatalytic synthesis of high-value-added compounds. [15,16] In current methods for synthesizing tryptophan-containing diketopiperazine heterodimers via P450 dimerase catalysis, the resulting DTDKPs are also limited due to the restricted substrate range of P450 dimerase. P450 dimerase has two substrate-binding pockets that accommodate the upper and lower TDKP portions, respectively. The lower pocket is relatively flexible, accepting a range of cWXs (where X is one of 20 natural amino acids) substrates, including cWA, cWP, cWV, cWI, cWL, cWM, cWF, cWY, and cWW, while the upper pocket is more limited. [7] Currently reported P450 dimerases include AspB, NasB, NascB, and Nas F5053 NzeB only accepts cWA, cWV, and cWP. [7,17–20] Nas F5053 Through enzyme engineering, its upper pocket can accept cWF, cWI, and cWL.

[21] However, it is still unacceptable to use substrates with greater spatial steric hindrance, such as cWW and cWY, and the substrate range remains limited, which greatly restricts the structural diversity of DTDKPs.

[0005] Therefore, there is an urgent need to provide a new biosynthetic method for diketopiperazine heterodimers to address the shortcomings of existing technologies. Summary of the Invention

[0006] The purpose of this invention is to provide a diketopiperazine heterodimer, its biosynthetic method, and its applications. In this invention, the inventors first discovered a P450 dimerase capable of producing natural diketopiperazine heterodimer products. Then, through site-directed mutagenesis, the P450 dimerase was modified to obtain a P450 dimerase mutant with significantly enhanced catalytic activity. This P450 dimerase or its mutant was then used to catalyze the biosynthesis of diketopiperazine containing tryptophan into diketopiperazine heterodimers with different regio / stereoselectivity. This biosynthetic method is green, environmentally friendly, highly efficient, and greatly expands the variety of diketopiperazine heterodimers.

[0007] In a first aspect, the present invention provides a method for the biosynthesis of diketopiperazine heterodimers, comprising the following steps: S1, culturing recombinant cells containing P450 dimerase or its mutant, and obtaining whole-cell cultures after induction of expression; S2, adding a tryptophan-containing cyclic dipeptide substrate to the whole-cell cultures and performing a catalytic reaction, and obtaining diketopiperazine heterodimers after separation and purification of the product.

[0008] In this invention, the inventors discovered that obtaining diketopiperazine heterodimers through biosynthesis has the advantages of being green and environmentally friendly, highly efficient, and greatly expanding the variety of diketopiperazine heterodimers.

[0009] In some embodiments, step S1 involves the following method for preparing recombinant cells containing P450 dimerase or its mutant: amplifying a nucleic acid molecule encoding P450 dimerase or its mutant, and ligating it to an expression vector to obtain a recombinant vector; transforming the recombinant vector into host cells to obtain recombinant cells containing P450 dimerase or its mutant; wherein the expression vector is pPWW50A vector and / or pMV261 vector, and the host cell is *Streptomyces cerevisiae* M1146 and / or *Mycobacterium smegmatis* MC. 2 155.

[0010] In some embodiments, the P450 dimerase or its mutants are selected from any of the following: A1) AcolP450 dimerase with the amino acid sequence shown in SEQ ID NO: 1, or a protein having more than 90% sequence identity and the same function as the amino acid sequence shown in SEQ ID NO: 1; A2) AchaP450 dimerase with the amino acid sequence shown in SEQ ID NO: 2, or a protein having more than 90% sequence identity and the same function as the amino acid sequence shown in SEQ ID NO: 2; A3) StsuP450 dimerase with the amino acid sequence shown in SEQ ID NO: 3, or a protein having more than 90% sequence identity and the same function as the amino acid sequence shown in SEQ ID NO: 3; A4) StnoP450 dimerase with the amino acid sequence shown in SEQ ID NO: 4, or a protein having more than 90% sequence identity and the same function as the amino acid sequence shown in SEQ ID NO: 4; A5) A… A5) The StsuP450-A398F mutant shown in SEQ ID NO: 5, or a protein with more than 90% sequence identity and the same function as the amino acid sequence shown in SEQ ID NO: 6; A6) The StsuP450-A398L mutant with the amino acid sequence shown in SEQ ID NO: 6, or a protein with more than 90% sequence identity and the same function as the amino acid sequence shown in SEQ ID NO: 6; A7) The StsuP450-W184F mutant with the amino acid sequence shown in SEQ ID NO: 7, or a protein with more than 90% sequence identity and the same function as the amino acid sequence shown in SEQ ID NO: 7; A8) The StsuP450-W184L mutant with the amino acid sequence shown in SEQ ID NO: 8, or a protein with more than 90% sequence identity and the same function as the amino acid sequence shown in SEQ ID NO: 8; A9) The StsuP450-W184A mutant with the amino acid sequence shown in SEQ ID NO: 9, or a protein with more than 90% sequence identity and the same function as the amino acid sequence shown in SEQ ID NO: 9. The amino acid sequence shown in Figure 9 has more than 90% sequence identity and the same function.

[0011] The P450 dimerase or its mutant provided by the present invention can be a natural, recombinant or synthetic active polypeptide. The active polypeptide can be a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic host (e.g., Escherichia coli) or a eukaryotic host (e.g., yeast, higher plants) using recombinant technology.

[0012] In some embodiments, the nucleic acid molecule encoding the P450 dimerase or a mutant thereof is selected from any of the following: B1) a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 10, or a nucleic acid molecule that hybridizes under stringent conditions with the nucleic acid molecule defined in B1) and encodes the aforementioned AcolP450 dimerase, or a nucleic acid molecule that has more than 90% sequence identity with the nucleic acid molecule defined in B1) and encodes the aforementioned AcolP450 dimerase; B2) a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 11, or a nucleic acid molecule that hybridizes under stringent conditions with the nucleic acid molecule defined in B2) and encodes the aforementioned AchaP450 dimerase, or a nucleic acid molecule that has more than 90% sequence identity with the nucleic acid molecule defined in B2) and encodes the aforementioned AchaP450 dimerase; B3) a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 10, or a nucleic acid molecule that hybridizes under stringent conditions with the nucleic acid molecule defined in B2) and encodes the aforementioned AchaP450 dimerase; Nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO: 12, or nucleic acid molecules that hybridize under stringent conditions with the nucleic acid molecule defined in B3) and encode the aforementioned StsuP450 dimerase, or nucleic acid molecules that have more than 90% sequence identity with the nucleic acid molecule defined in B3) and encode the aforementioned StsuP450 dimerase; B4) nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO: 13, or nucleic acid molecules that hybridize under stringent conditions with the nucleic acid molecule defined in B4) and encode the aforementioned StnoP450 dimerase, or nucleic acid molecules that have more than 90% sequence identity with the nucleic acid molecule defined in B4) and encode the aforementioned StnoP450 dimerase; B5) nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO: Nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO: 14, or nucleic acid molecules that hybridize under stringent conditions with the nucleic acid molecule defined in B5) and encode the aforementioned StsuP450-A398F mutant, or nucleic acid molecules that have more than 90% sequence identity with the nucleic acid molecule defined in B5) and encode the aforementioned StsuP450-A398F mutant; B6) Nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO: 15, or nucleic acid molecules that hybridize under stringent conditions with the nucleic acid molecule defined in B6) and encode the aforementioned StsuP450-A398L mutant, or nucleic acid molecules that have more than 90% sequence identity with the nucleic acid molecule defined in B6) and encode the aforementioned StsuP450-A398L mutant; B7) Nucleic acid molecules having the nucleotide sequence shown in SEQ ID NO: Nucleic acid molecules with the nucleotide sequence shown in 16, or nucleic acid molecules that hybridize with the nucleic acid molecules defined in B7) under strict conditions and encode the above-mentioned StsuP450-W184F mutant, or nucleic acid molecules that have more than 90% sequence identity with the nucleic acid molecules defined in B7) and encode the above-mentioned StsuP450-W184F mutant.B8) A nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 17, or a nucleic acid molecule that hybridizes under stringent conditions with the nucleic acid molecule defined in B8) and encodes the StsuP450-W184L mutant, or a nucleic acid molecule that has more than 90% sequence identity with the nucleic acid molecule defined in B8) and encodes the StsuP450-W184L mutant; B9) A nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO: 18, or a nucleic acid molecule that hybridizes under stringent conditions with the nucleic acid molecule defined in B9) and encodes the StsuP450-W184A mutant, or a nucleic acid molecule that has more than 90% sequence identity with the nucleic acid molecule defined in B9) and encodes the StsuP450-W184A mutant.

[0013] As used herein, the term "hybridization under stringent conditions" refers to the hybridization of two nucleic acid fragments under standard hybridization conditions as described in the section "Expression of Cloned Genes in E. coli" of *Molecular Cloning: A Laboratory Manual* (1989) (Cold Spring Lane Laboratory, New York, USA). Such conditions include hybridization in 6.0 × SSC at 45 °C, followed by washing in 2 × SSC at 50 °C. To select stringency, the salt concentration in the washing step can be chosen, for example, between 2.0 × SSC at 50 °C for low stringency and 2.0 × SSC at 50 °C for high stringency. Additionally, the temperature in the washing step can be varied between room temperature (approximately 22 °C) for low stringency and 65 °C for high stringency.

[0014] As used herein, the term “sequence identity” can be evaluated by the naked eye or by computer software (such as the software program described in Current Protocols in Molecular Biology by Ausubel et al. eds. (2007)). Molecules are identical at that position when positions in the compared sequences are occupied by the same bases or amino acids. Identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. “Sequence identity” of a polynucleotide or amino acid sequence with another sequence having a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that when the sequences are aligned, that percentage of bases or amino acids are the same in the two compared sequences.

[0015] The nucleic acid molecules provided by this invention can usually be obtained by PCR amplification or artificial synthesis.

[0016] In some implementations, step S1, the induction of expression step specifically includes: when cultured to OD... 600When the value is 0.8-1.0, add 0.1-0.5% acetamide, 0.05-0.2% ALA, and 0.05-0.2% Fe(NH4)2(SO4)2, and induce for 12-16 h at a temperature of 25-30℃ and a rotation speed of 200-240 rpm.

[0017] In some embodiments, in step S2, the concentration of the tryptophan-containing cyclic dipeptide substrate is 80-120 μM, and the tryptophan-containing cyclic dipeptide substrate is selected from at least one of cWW, cWF, cWY, cWL, cWA, cWM, cWV, cWI, cWP, 5,5′-difluoro-cWW, 6,6′-difluoro-cWW, 6,6′-dichloro-cWW, and 6,6′-dibromo-cWW.

[0018] In this invention, cWW is a cyclic dipeptide containing tryptophan-tryptophan, cWF is a cyclic dipeptide containing tryptophan-phenylalanine, cWY is a cyclic dipeptide containing tryptophan-tyrosine, cWL is a cyclic dipeptide containing tryptophan-leucine, cWA is a cyclic dipeptide containing tryptophan-alanine, cWM is a cyclic dipeptide containing tryptophan-methionine, cWV is a cyclic dipeptide containing tryptophan-valine, cWI is a cyclic dipeptide containing tryptophan-isoleucine, cWP is a cyclic dipeptide containing tryptophan-proline, cWS is a cyclic dipeptide containing tryptophan-serine, cWG is a cyclic dipeptide containing tryptophan-glycine, 5,5′-difluoro-cWW is a cyclic dipeptide containing 5F-tryptophan-5′F-tryptophan, and 6,6′-difluoro-cWW is a cyclic dipeptide containing 6F-tryptophan-6′F-tryptophan. 6,6′-dichloro-cWW is a cyclic dipeptide containing 6Cl-tryptophan-6′Cl-tryptophan, and 6,6′-dibromo-cWW is a cyclic dipeptide containing 6Br-tryptophan-6′Br-tryptophan.

[0019] In some implementations, the catalytic reaction specifically includes reacting for 2-4 days at a temperature of 25-30°C and a rotation speed of 700-900 rpm.

[0020] In a second aspect, the present invention provides a diketopiperazine heterodimer synthesized by any of the above-described biosynthetic methods.

[0021] In some embodiments, the diketopiperazine heterodimer comprises the structure shown in Formulas 1-25 below: , , , , , 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。

[0022] In some preferred embodiments, the diketopiperazine heterodimer of Formula 1 is synthesized from cWW monosubstrate catalyzed by AcolP450 dimerase or AchaP450 dimerase; the diketopiperazine heterodimer of Formula 2 is synthesized from cWW monosubstrate catalyzed by AchaP450 dimerase or StsuP450 dimerase; the diketopiperazine heterodimer of Formula 3 is synthesized from cWW monosubstrate catalyzed by StnoP450 dimerase; the diketopiperazine heterodimer of Formula 4 is synthesized from cWY monosubstrate catalyzed by StsuP450 dimerase or StnoP450 dimerase; and the diketopiperazine heterodimer of Formula 5 is synthesized from cWY and cWF catalyzed by StsuP450 dimerase. The synthesis of diketopiperazine heterodimers is as follows: Formula 6 shows the synthesis of cWF and cWY disubstrates catalyzed by StsuP450 dimerase; Formula 7 shows the synthesis of cWF monosubstrate catalyzed by StsuP450 dimerase or StnoP450 dimerase; Formula 8 shows the synthesis of cWF monosubstrate catalyzed by AcolP450 dimerase; Formula 9 shows the synthesis of cWY monosubstrate catalyzed by AcolP450 dimerase or StnoP450 dimerase; Formula 10 shows the synthesis of cWY monosubstrate catalyzed by AcolP450 dimerase, StsuP450 dimerase, or StnoP450 dimerase. The synthesis of cWL monosubstrate is catalyzed by StnoP450 dimerizing enzyme; the diketopiperazine heterodimer shown in Formula 11 is synthesized from cWF monosubstrate catalyzed by StnoP450 dimerizing enzyme; the diketopiperazine heterodimer shown in Formula 12 is synthesized from cWY monosubstrate catalyzed by StnoP450 dimerizing enzyme; the diketopiperazine heterodimer shown in Formula 13 is synthesized from cWW and cWL bisubstrate catalyzed by AcolP450 dimerizing enzyme; the diketopiperazine heterodimer shown in Formula 14 is synthesized from cWF and cWV bisubstrate catalyzed by StsuP450 dimerizing enzyme; the diketopiperazine heterodimer shown in Formula 15 is synthesized from cWY and cWL bisubstrate catalyzed by StnoP450 dimerizing enzyme; the diketopiperazine heterodimer shown in Formula 16 is synthesized from StsuP450 dimerizing enzyme. The cWI monosubstrate was synthesized using P450 dimerase or the StsuP450-W184L mutant; the diketopiperazine heterodimer shown in Formula 17 was synthesized using 5,5′-difluoro-cWW monosubstrate catalyzed by StsuP450 dimerase; the diketopiperazine heterodimer shown in Formula 18 was synthesized using 5,5′-difluoro-cWW monosubstrate catalyzed by StsuP450 dimerase; the diketopiperazine heterodimer shown in Formula 19 was synthesized using 6,6′-difluoro-cWW monosubstrate catalyzed by StsuP450 dimerase; and the diketopiperazine heterodimer shown in Formula 20 was synthesized using cWA monosubstrate catalyzed by the StsuP450-A398F mutant.The diketopiperazine heterodimer shown in Formula 21 is synthesized from a single cWP substrate catalyzed by the StsuP450-A398F mutant; the diketopiperazine heterodimer shown in Formula 22 is synthesized from a dual cWW and cWA substrate catalyzed by the StsuP450-A398F mutant, the StsuP450-A398L mutant, the StsuP450-W184F mutant, the StsuP450-W184L mutant, or the StsuP450-W184A mutant; the diketopiperazine heterodimer shown in Formula 23 is synthesized from a single cWP substrate catalyzed by the StsuP450-A398F mutant, the StsuP450-A398L mutant, the StsuP450-W184F mutant, or the StsuP450-W184A mutant. The tsuP450-W184L mutant catalyzes the synthesis of cWW and cWP dual substrates; the diketopiperazine heterodimer shown in Formula 24 is synthesized from cWW and cWS dual substrates catalyzed by the StsuP450-A398F mutant, the StsuP450-A398L mutant, the StsuP450-W184F mutant, or the StsuP450-W184L mutant; the diketopiperazine heterodimer shown in Formula 25 is synthesized from cWW and cWG dual substrates catalyzed by the StsuP450-A398F mutant, the StsuP450-A398L mutant, the StsuP450-W184F mutant, or the StsuP450-W184L mutant.

[0023] In a third aspect, the present invention provides the use of the diketopiperazine heterodimer as described above in the preparation of anti-inflammatory, anticancer, antimalarial, and antiviral drugs.

[0024] The beneficial effects of this invention are as follows: Unlike existing technologies, the P450 TDKP dimerase of this invention possesses unique substrate specificity—it can accept large TDKP monomers as substrates, and the mutants obtained through rational mutation further expand the substrate spectrum to simultaneously accept both large and small substrates (including novel substrates cWS and cWG). P450 or its mutants expand the substrate spectrum of TDKP dimerase, catalyzing the synthesis of diketopiperazine heterodimers with different regio / stereoselectivities. This biosynthetic method is green, environmentally friendly, highly efficient, and greatly expands the diversity of diketopiperazine heterodimers. Attached Figure Description

[0025] Figure 1 The images show the liquid chromatography results of the products of CDPS enzyme and P450 dimerase expressed in Streptomyces cerevisiae M1146 in Example 1 of this invention. Among them, (a) is the liquid chromatography result of AcolP450 dimerase, (b) is the liquid chromatography result of AchaP450 dimerase, (c) is the liquid chromatography result of StnoP450 dimerase, and (d) is the liquid chromatography result of StsuP450 dimerase. Figure 2 Compound 1 in Example 1 of this invention 1 H NMR spectrum; Figure 3 Compound 2 in Example 1 of this invention 1 H NMR spectrum; Figure 4 Compound 3 in Example 1 of this invention 1 H NMR spectrum; Figure 5 The images shown are liquid phase detection results of whole-cell catalysis of single substrate dimer formation by Mycobacterium smegmatis containing AcolP450 dimerase in Example 2 of the present invention. Among them, (a) is the liquid phase detection result of cWW dimer formation, (b) is the liquid phase detection result of cWF dimer formation, (c) is the liquid phase detection result of cWY dimer formation, and (d) is the liquid phase detection result of cWL dimer formation. Figure 6 The images shown are liquid phase detection results of whole-cell catalysis of single substrate dimer formation by Mycobacterium smegmatis containing StsuP450 dimerase in Example 2 of this invention. Among them, (a) is the liquid phase detection result of cWW dimer formation, (b) is the liquid phase detection result of cWF dimer formation, (c) is the liquid phase detection result of cWY dimer formation, (d) is the liquid phase detection result of cWI dimer formation, (e) is the liquid phase detection result of cWL dimer formation, (f) is the liquid phase detection result of 5,5′-difluoro-cWW dimer formation, and (g) is the liquid phase detection result of 6,6′-difluoro-cWW dimer formation. Figure 7 The images shown are liquid phase detection results of whole-cell catalysis of single substrate dimer formation by Mycobacterium smegmatis containing StnoP450 dimerase in Example 2 of the present invention. Among them, (a) is the liquid phase detection result of cWW dimer formation, (b) is the liquid phase detection result of cWF dimer formation, (c) is the liquid phase detection result of cWY dimer formation, and (d) is the liquid phase detection result of cWL dimer formation. Figure 8A The above are liquid phase detection results of whole-cell catalysis of two substrates to form dimers by Mycobacterium smegmatis containing AcolP450 dimerase in Example 3 of the present invention. Among them, (a) is the liquid phase detection result of catalysis of cWW and cWF to form dimers, (b) is the liquid phase detection result of catalysis of cWW and cWY to form dimers, (c) is the liquid phase detection result of catalysis of cWW and cWL to form dimers, and (d) is the liquid phase detection result of catalysis of cWW and cWA to form dimers. Figure 8BThe above are liquid phase detection results of whole-cell catalysis of two substrates to form dimers by Mycobacterium smegmatis containing AcolP450 dimerase in Example 3 of the present invention. Among them, (a) is the liquid phase detection result of catalysis of cWF and cWY to form dimers, (b) is the liquid phase detection result of catalysis of cWF and cWL to form dimers, and (c) is the liquid phase detection result of catalysis of cWF and cWA to form dimers. Figure 8C The above are liquid phase detection results of whole-cell catalysis of two substrates to form dimers by Mycobacterium smegmatis containing AcolP450 dimerase in Example 3 of the present invention. Among them, (a) is the liquid phase detection result of catalysis of cWY and cWL to form dimers, and (b) is the liquid phase detection result of catalysis of cWY and cWM to form dimers. Figure 8D This is a liquid phase detection result of the formation of dimers from cWL and cWA by whole cells of Mycobacterium smegmatis containing AcolP450 dimerase in Example 3 of the present invention; Figure 9A The above are liquid phase detection results of whole-cell catalysis of bisubstrate dimer formation by *Mycobacterium smegmatis* containing StsuP450 dimerase in Example 3 of this invention. (i) shows the liquid phase detection results of dimer formation by cWW and cWF; (ii) shows the liquid phase detection results of dimer formation by cWW and cWY; (iii) shows the liquid phase detection results of dimer formation by cWW and cWL; (iv) shows the liquid phase detection results of dimer formation by cWW and cWV; and (v) shows the liquid phase detection results of dimer formation by cWW and cWI. The liquid phase detection results of the dimer are shown in (vi), (vii), (vii), (viii), (viii), and (ix). Figure 9BThe above are liquid phase detection results of whole-cell catalysis of bisubstrate dimer formation by *Mycobacterium smegmatis* containing StsuP450 dimerase in Example 3 of this invention. (i) shows the liquid phase detection results of dimer formation catalyzed by cWY and cWF; (ii) shows the liquid phase detection results of dimer formation catalyzed by cWY and cWL; (iii) shows the liquid phase detection results of dimer formation catalyzed by cWY and cWM; (iv) shows the liquid phase detection results of dimer formation catalyzed by cWY and cWV; (v) shows the liquid phase detection results of dimer formation catalyzed by cWY and cWP; (v... i) is the liquid phase detection result of catalyzing the formation of dimers from cWY and cWI; (vii) is the liquid phase detection result of catalyzing the formation of dimers from cWY and 5,5′-difluoro-cWW; (viii) is the liquid phase detection result of catalyzing the formation of dimers from cWY and 6,6′-difluoro-cWW; (ix) is the liquid phase detection result of catalyzing the formation of dimers from cWY and 6,6′-dichloro-cWW; and (x) is the liquid phase detection result of catalyzing the formation of dimers from cWY and 6,6′-dibromo-cWW. Figure 9C The above are liquid phase detection results of whole-cell catalysis of bisubstrate dimer formation by *Mycobacterium smegmatis* containing StsuP450 dimerase in Example 3 of this invention. (i) shows the liquid phase detection results of dimer formation by cWF and cWL; (ii) shows the liquid phase detection results of dimer formation by cWF and cWM; (iii) shows the liquid phase detection results of dimer formation by cWF and cWV; (iv) shows the liquid phase detection results of dimer formation by cWF and cWI; and (v) shows the liquid phase detection results of dimer formation by cWF and 5,5′-difluoro-cWW. The liquid phase detection results of dimer formation are shown in (vi), (vii), and (viii). Figure 9DThe above are liquid phase detection results of whole-cell catalytic bisubstrate dimer formation by *Mycobacterium smegmatis* containing StsuP450 dimerase in Example 3 of this invention. (i) shows the liquid phase detection results of dimer formation catalyzed by cWI and cWL; (ii) shows the liquid phase detection results of dimer formation catalyzed by cWI and cWM; (iii) shows the liquid phase detection results of dimer formation catalyzed by cWI and cWV; (iv) shows the liquid phase detection results of dimer formation catalyzed by cWI and cWS; and (v) shows the liquid phase detection results of dimer formation catalyzed by cWI and cWA. The liquid phase detection results of the dimer are shown in (vi), (vii), (vii), (viii), (viii), and (ix). Figure 9E The above are liquid phase detection results of the whole-cell catalysis of the formation of two substrate dimers by Mycobacterium smegmatis containing StsuP450 dimerase in Example 3 of the present invention. Among them, (i) is the liquid phase detection result of the formation of dimers by cWL and cWM, (ii) is the liquid phase detection result of the formation of dimers by cWL and cWV, (iii) is the liquid phase detection result of the formation of dimers by cWL and 5,5′-difluoro-cWW, (iv) is the liquid phase detection result of the formation of dimers by cWL and 6,6′-difluoro-cWW, (v) is the liquid phase detection result of the formation of dimers by cWL and 6,6′-dichloro-cWW, and (vi) is the liquid phase detection result of the formation of dimers by cWL and 6,6′-dibromo-cWW. Figure 9F The above are liquid phase detection results of the whole-cell catalysis of the formation of a two-substrate dimer by *Mycobacterium smegmatis* containing StsuP450 dimerase in Example 3 of the present invention. (i) shows the liquid phase detection results of the formation of a dimer by catalyzing 5,5′-difluoro-cWV and cWV, and (ii) shows the liquid phase detection results of the formation of a dimer by catalyzing 6,6′-difluoro-cWW and cWV. Figure 10AThe above are liquid phase detection results of whole-cell catalysis of two substrates to form dimers by Mycobacterium smegmatis containing StnoP450 dimerase in Example 3 of the present invention. Among them, (i) is the liquid phase detection result of catalysis of cWW and cWF to form dimers, (ii) is the liquid phase detection result of catalysis of cWW and cWL to form dimers, (iii) is the liquid phase detection result of catalysis of cWW and cWA to form dimers, and (iv) is the liquid phase detection result of catalysis of cWW and cWV to form dimers. Figure 10B The above are liquid phase detection results of whole-cell catalysis of two substrates to form dimers by Mycobacterium smegmatis containing StnoP450 dimerase in Example 3 of the present invention. Among them, (i) is the liquid phase detection result of catalysis of cWF and cWL to form dimers, (ii) is the liquid phase detection result of catalysis of cWF and cWA to form dimers, (iii) is the liquid phase detection result of catalysis of cWF and cWI to form dimers, (iv) is the liquid phase detection result of catalysis of cWF and cWV to form dimers, and (v) is the liquid phase detection result of catalysis of cWF and cWY to form dimers. Figure 10C The above are liquid phase detection results of the whole-cell catalysis of the formation of a two-substrate dimer by Mycobacterium smegmatis containing StnoP450 dimerase in Example 3 of the present invention. Among them, (i) is the liquid phase detection result of the formation of a dimer by cWY and cWL, (ii) is the liquid phase detection result of the formation of a dimer by cWY and cWV, (iii) is the liquid phase detection result of the formation of a dimer by cWY and cWI, and (iv) is the liquid phase detection result of the formation of a dimer by cWY and cWA. Figure 10D The above are liquid phase detection results of the whole-cell catalysis of the formation of a two-substrate dimer by Mycobacterium smegmatis containing StnoP450 dimerase in Example 3 of the present invention. Among them, (i) is the liquid phase detection result of the formation of a dimer by cWL and cWA, and (ii) is the liquid phase detection result of the formation of a dimer by cWL and cWV. Figure 11A The above are liquid phase detection results of whole-cell catalysis of single substrate dimer formation by Mycobacterium smegmatis containing the StsuP450-A398F mutant in Example 2 of the present invention, wherein (i) is the liquid phase detection result of cWA dimer formation and (ii) is the liquid phase detection result of cWP dimer formation; Figure 11BThe above are liquid phase detection results of whole-cell catalysis of two substrates to form dimers by Mycobacterium smegmatis containing StsuP450-A398F and StsuP450-A398L in Example 3 of the present invention. Among them, (i) is the liquid phase detection result of catalysis of cWW and cWA to form dimers, (ii) is the liquid phase detection result of catalysis of cWW and cWP to form dimers, (iii) is the liquid phase detection result of catalysis of cWW and cWS to form dimers, and (iv) is the liquid phase detection result of catalysis of cWW and cWG to form dimers. Figure 11C The above are liquid phase detection results of whole-cell catalysis of two substrates to form dimers by Mycobacterium smegmatis containing StsuP450-W184F, StsuP450-W184L and StsuP450-W184A in Example 3 of the present invention. Among them, (i) is the liquid phase detection result of catalysis of cWW and cWA to form dimers, (ii) is the liquid phase detection result of catalysis of cWW and cWP to form dimers, (iii) is the liquid phase detection result of catalysis of cWW and cWS to form dimers, and (iv) is the liquid phase detection result of catalysis of cWW and cWG to form dimers. Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0028] Example 1: Molecular cloning and heterologous expression of the AcolP450, AchaP450, StnoP450, and StsuP450 genes. 1.1 Obtaining the genes encoding AcolP450, AchaP450, StnoP450, and StsuP450 dimerases Through genome mining, gene clusters containing both CDPS and P450 genes were identified, namely Acol (containing CDPS and P450 genes), Acha (containing CDPS and P450 genes), Stsu (containing CDPS and P450 genes), and Stno (containing CDPS and P450 genes).

[0029] Specifically, the AcolCDPS and AcolP450 genes originate from Streptomyces ( Actinomadura oligospora The nucleotide sequences of the two genes are shown in SEQ ID NO: 19 and SEQ ID NO: 10, respectively, and the genes were synthesized at Wuhan Jinkairui Biotechnology Co., Ltd.; the AchaCDPS and AchaP450 genes were derived from Streptomyces ( Actinomadura harenae The nucleotide sequences of the gene are shown in SEQ ID NO: 20 and SEQ ID NO: 11, respectively, and the gene was synthesized at Wuhan Jinkairui Biotechnology Co., Ltd.; the StsuCDPS and StsuP450 genes were derived from Streptomyces ( Streptomyces sulfonofaciens The nucleotide sequences of the two genes are shown in SEQ ID NO: 21 and SEQ ID NO: 12, respectively, and the genes were synthesized at Wuhan Jinkairui Biotechnology Co., Ltd.; the StnoCDPS and StnoP450 genes were derived from Streptomyces ( Streptomyces noursei The nucleotide sequences of the gene are shown in SEQ ID NO: 22 and SEQ ID NO: 13, respectively, and the gene was synthesized at Wuhan Jinkairui Biotechnology Co., Ltd.

[0030] 1.2 Construction of Streptomyces expression vectors encoding AcolP450, AchaP450, StnoP450, and StsuP450 dimerases DNA fragments encoding CDPS and P450 enzymes (the synthesized AcolCDPS and AcolP450 gene fragments, AchaCDPS and AchaP450 gene fragments, StsuCDPS and StsuP450 gene fragments, and StnoCDPS and StnoP450 gene fragments) were amplified using conventional PCR techniques. These fragments were then ligated into the pPWW50A vector to obtain the plasmids pPWW50A-Acol(CDPS+P450), pPWW50A-Acha(CDPS+P450), pPWW50A-Stsu(CDPS+P450), and pPWW50A-Stno(CDPS+P450). These plasmids were then transformed into... E. coliDH5α competent cells were seeded in a medium containing 50 The samples were placed on LB solid medium containing μg / mL apramycin. Positive transformants were selected and confirmed by DNA sequencing. Plasmids were extracted from the correctly sequenced positive transformants to obtain the following plasmids: pPWW50A-AcolCDPS-AcolP450, pPWW50A-AchaCDPS-AchaP450, pPWW50A-StsuCDPS-StsuP450, and pPWW50A-StnoCDPS-StnoP450. These plasmids were then transformed into *Streptomyces cerevisiae* M1146 via conjugation transfer to obtain *Streptomyces cerevisiae* M1146 / pPWW50A-Acol(CDPS+P450), M1146 / pPWW50A-Acha(CDPS+P450), M1146 / pPWW50A-Stsu(CDPS+P450), and M1146 / pPWW50A-Stno(CDPS+P450).

[0031] 1.3 Obtaining natural products of AcolP450, AchaP450, StnoP450, and StsuP450 dimerizing enzymes Following the method in step 1.2, the following strains of *Streptomyces cerevisiae* were constructed: M1146 / pPWW50A-Acol(CDPS+P450), M1146 / pPWW50A-Acha(CDPS+P450), M1146 / pPWW50A-Stsu(CDPS+P450), M1146 / pPWW50A-Stno(CDPS+P450), and *Streptomyces cerevisiae* M1146 / pPWW50A-AcolCDPS, M1146 / pPWW50A-AchaCDPS, M1146 / pPWW50A-StsuCDPS, and M1146 / pPWW50A-StnoCDPS. Spores of these *Streptomyces cerevisiae* strains were then inoculated onto MS plates (50 μL). The spores were activated with 50 μg / mL apramycin and cultured at 28°C for about 7 days. The activated spores were then collected for liquid culture. The activated spores were inoculated into GYM (50 μg / mL apramycin) medium and fermented at 28°C and 220 rpm for about 7 days. The fermentation broth was then extracted three times with equal volumes of ethyl acetate. The upper organic phase was collected, evaporated to dryness, and then preliminarily separated and purified by silica gel column chromatography. Further separation and purification were performed by semi-preparative liquid chromatography. The purified product was first detected by high performance liquid chromatography and then identified by NMR to obtain the product structure.

[0032] The high-performance liquid chromatography (HPLC) detection conditions are as follows: an Agilent SB-C18 column was used, the organic phase was acetonitrile, the aqueous phase was ultrapure water containing 0.1% formic acid, and the injection volume was 10 μL; the method was 10%-60% acetonitrile for 20 min, 60%-100% acetonitrile for 1 min, 100% acetonitrile for 4 min, and 10% acetonitrile for 5 min, the flow rate was 1 mL / min, and the single sample analysis time was 30 min.

[0033] Chromatograms detected by high performance liquid chromatography are as follows: Figure 1 As shown. From Figure 1 As can be seen, expression of AcolCDPS, AchaCDPS, and StnoCDPS can yield cWW, while expression of StsuCDPS can yield cWY and cWF. Furthermore, expression of AcolP450 can catalyze the reaction of cWW to synthesize compound 1, expression of AchaP450 can catalyze the reaction of cWW to synthesize compounds 1 and 2, expression of StnoP450 can catalyze the reaction of cWW to synthesize compound 3, and expression of StsuP450 can catalyze the reaction of cWY and cWW to synthesize compounds 4, 5, 6, and 7, respectively.

[0034] For example, the NMR spectrum of compound 1 is as follows: Figure 2 As shown, the NMR spectrum of compound 2 is as follows: Figure 3 As shown, the NMR spectrum of compound 3 is as follows: Figure 4 As shown.

[0035] For example, the HR-EI-MS data of compounds 1, 2, and 3 are shown in Table 1 below, and the NMR data of compounds 1, 2, and 3 are shown in Tables 2-4.

[0036] Table 1. HR-EI-MS data of compounds 1, 2, and 3

[0037] Table 2 NMR data of compound 1

[0038]

[0039] Table 3 NMR data of compound 2

[0040]

[0041] Table 4 NMR data of compound 3

[0042]

[0043] Similarly, the structural formulas of compounds 4-7 were obtained by NMR identification, as shown below: , , , .

[0044] In summary, the AcolP450, AchaP450, StsuP450, and StnoP450 dimerizing enzymes provided by this invention have the activity of catalyzing the reaction of tryptophan-containing cyclic dipeptides to generate diketopiperazine heterodimers.

[0045] Example 2: AcolP450, StnoP450, StsuP450 dimerases or StsuP450 dimerase mutants catalyze the self-dimerization of monocyclic dipeptide substrates. 2.1 Construction of Mycobacterium smegmatis expression vector encoding AcolP450, StnoP450, StsuP450 dimerase or StsuP450 dimerase mutant DNA fragments encoding the P450 enzyme (the synthesized AcolP450, StnoP450, and StsuP450 gene fragments) were amplified using conventional PCR techniques. These fragments were then ligated into the pMV261 vector, and subsequently plasmidized and transformed into... E. coli DH5α competent cells were spread on LB solid medium containing kanamycin; positive transformants were selected and confirmed by DNA sequencing to obtain pMV261-AcolP450 plasmid, pMV261-StnoP450 plasmid, and pMV261-StsuP450 plasmid. Using the pMV261-StsuP450 plasmid as a template, plasmids containing the mutants StsuP450-A398F, StsuP450-A398L, StsuP450-W184F, StsuP450-W184L, and StsuP450-W184A were obtained through site-directed mutagenesis. These plasmids were then transformed into *Mycobacterium smegmaecum* MC via electroporation. 2 In 155, recombinant Mycobacterium smegmae MC was obtained. 2 155 / pMV261-AcolP450, MC 2 155 / pMV261-StnoP450, MC 2155 / pMV261-StsuP450, MC 2 155 / pMV261-StsuP450-A398F, MC 2 155 / pMV261-StsuP450-A398L, MC 2 155 / pMV261-StsuP450-W184F, MC 2 155 / pMV261-StsuP450-W184L, MC 2 155 / pMV261-StsuP450-W184A.

[0046] 2.2 Whole-cell catalysis of single substrate to produce dimer products by Mycobacterium smegmatis containing AcolP450, StsuP450, StnoP450 dimerase or a StsuP450 dimerase mutant. The recombinant strains obtained in section 2.1 were streaked onto LBG plates and incubated at 37°C for 3 days. Single colonies were picked and transferred to 2 mL of LBG containing kanamycin, and incubated at 37°C and 220 rpm for 2 days. The colonies were then transferred to 50 mL of LBG and incubated at 37°C and 220 rpm. When the OD... 600 When the pH value reached 0.8-1.0, 0.3% acetamide, 0.1% ALA, and 0.1% Fe(NH4)2(SO4)2 were added. After induction at 28℃ and 220 rpm for 14 h, the bacterial culture was transferred to a 24-well plate, and 100 μM of tryptophan-containing cyclic dipeptide substrates (cWW, cWF, cWI, cWY, cWL, 5,5′-difluoro-cWW, 6,6′-difluoro-cWW) were added. The reaction was carried out at 28℃ and 800 rpm for 3 days. The bacterial culture was extracted three times with an equal volume of ethyl acetate, and the upper organic phase was collected. After the organic phase was evaporated to dryness, a portion was dissolved in methanol and the reaction product was detected by high performance liquid chromatography (detection conditions as shown in Example 1). The other portion was initially separated and purified by silica gel column chromatography, and then further separated and purified by semi-preparative liquid chromatography to obtain the purified product.

[0047] The chromatograms for high performance liquid chromatography detection are as follows: Figure 5-7 and Figure 11A As shown.

[0048] from Figure 5 It can be seen that the whole cells of Mycobacterium smegmatis containing AcolP450 dimerase catalyze the cWW reaction to synthesize compound 1, the cWF reaction to synthesize compound 8, the cWY reaction to synthesize compound 9, and the cWL reaction to synthesize compound 10.

[0049] from Figure 6 As can be seen, the whole cells of Mycobacterium smegmatis containing StsuP450 dimerase catalyze the cWW reaction to synthesize compound 2, the cWF reaction to synthesize compound 7, the cWY reaction to synthesize compound 4, the cWI reaction to synthesize compound 16, the cWL reaction to synthesize compound 10, the 5,5′-difluoro-cWW reaction to synthesize compounds 17 and 18, and the 6,6′-difluoro-cWW reaction to synthesize compound 19.

[0050] from Figure 7 It can be seen that whole-cell mycobacteria containing StnoP450 dimerase catalyze the cWW reaction to synthesize compound 3, catalyze the cWF reaction to synthesize compounds 7 and 11, catalyze the cWY reaction to synthesize compounds 4, 9 and 12, and catalyze the cWL reaction to synthesize compound 10.

[0051] from Figure 11A It can be seen that whole-cell mycobacteria containing StsuP450-A398F catalyze the cWA reaction to synthesize compound Naseseazine D20, and catalyze the cWP reaction to synthesize compound Naseseazine B21.

[0052] Furthermore, the structural formulas of compounds 8-12 and 16-21 were obtained by LC-MS comparison and NMR identification, as shown below: , , , , , , , , , , .

[0053] Example 3: AcolP450, StnoP450, StsuP450 dimerases or StsuP450 dimerase mutants catalyze cross-dimerization of bicyclic dipeptide substrates. 3.1 Whole-cell catalysis of bisubstrate by Mycobacterium smegmatis containing AcolP450, StsuP450, StnoP450 dimerase or a StsuP450 dimerase mutant to produce dimer products. The recombinant strains obtained in section 2.1 were streaked onto LBG plates and incubated at 37°C for 3 days. Single colonies were picked and transferred to 2 mL of LBG containing kanamycin, and incubated at 37°C and 220 rpm for 2 days. The colonies were then transferred to 50 mL of LBG and incubated at 37°C and 220 rpm. When the OD... 600When the pH value reached 0.8-1.0, 0.3% acetamide, 0.1% ALA, and 0.1% Fe(NH4)2(SO4)2 were added. After induction at 28℃ and 220 rpm for 14 h, the bacterial culture was transferred to 24-well plates. 100 μM of tryptophan-containing cyclic dipeptide substrates (cWW, cWF, cWI, cWY, cWL, 5,5′-difluoro-cWW, 6,6′-difluoro-cWW) and 100 μM of other tryptophan-containing cyclic dipeptide substrates (cWF, cWY, cWL, cWA, cWM, cWV, cWI, cWP, 5,5′-difluoro-cWW, 6,6′-difluoro-cWW, 6,6′-dichloro-cWW, 6,6′-dibromo-cWW) were added. The culture was then incubated at 28℃ and 800 rpm. The reaction was carried out at rpm for 3 days. The bacterial culture was extracted three times with an equal volume of ethyl acetate. The upper organic phase was collected. After the organic phase was evaporated to dryness, a portion was dissolved in methanol and the reaction product was detected by high performance liquid chromatography (detection conditions as shown in Example 1). The other portion was initially separated and purified by silica gel column chromatography, and then further separated and purified by semi-preparative liquid chromatography to obtain the purified product.

[0054] The chromatograms for high performance liquid chromatography detection are as follows: Figures 8A-8D As shown in 9A-9F, 10A-10D, 11B-11C.

[0055] from Figures 8A-8D It can be seen that whole-cell mycobacterium smegmatis containing AcolP450 dimerase catalyzes the reaction of cWW and cWL to synthesize compound 13, as well as other structurally diverse diketopiperazine heterodimers.

[0056] from Figures 9A-9F It can be seen that whole-cell mycobacterium smegmatis containing StsuP450 dimerase catalyzes the reaction of cWF and cWV to synthesize compound 14, as well as other structurally diverse diketopiperazine heterodimers.

[0057] from Figure 10A-10D It can be seen that whole-cell mycobacterium smegmatis containing StnoP450 dimerase catalyzes the reaction of cWY and cWL to synthesize compound 15, as well as other structurally diverse diketopiperazine heterodimers.

[0058] from Figure 11B-11CAs can be seen, Mycobacterium smegmatis containing mutants such as StsuP450-A398F synthesized compound NAS-3822 by whole-cell catalysis of cWW and cWA, compound NAS-3523 by whole-cell catalysis of cWW and cWP, compound 24 by whole-cell catalysis of cWW and cWS, compound 25 by whole-cell catalysis of cWW and cWG, and other diketopiperazine heterodimers with diverse structures.

[0059] Furthermore, through LC-MS analysis, comparison with known products by HPLC, and NMR identification, the structural formulas of compounds 13-15 and 22-25 were obtained, as shown below: , , , , , , .

[0060] In summary, this invention utilizes the discovered P450 dimerase or its mutants to catalyze the biosynthesis of novel diketopiperazine heterodimers containing tryptophan with different regio / stereoselectivity. This biosynthesis method is green, environmentally friendly, highly efficient, and greatly expands the variety of diketopiperazine heterodimers.

[0061] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0062] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

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Claims

1. A method of biosynthesis of diketopiperazine heterodimers, characterized in that, The method comprises the following steps: S1. Culturing recombinant cells containing P450 dimerization enzyme or mutants thereof, obtaining whole cell culture after induced expression; S2. Adding cyclic dipeptide substrate containing tryptophan to the whole cell culture, and then performing catalytic reaction, and obtaining diketopiperazine isodimer after separation and purification.

2. The biosynthetic method according to claim 1, characterized in that, In step S1, the preparation method of the recombinant cells containing P450 dimerization enzyme or mutants thereof is as follows: amplifying nucleic acid molecules encoding the P450 dimerization enzyme or mutants thereof, and then linking the nucleic acid molecules with an expression vector to obtain a recombinant vector; and transforming the recombinant vector into host cells to obtain recombinant cells containing P450 dimerization enzyme or mutants thereof; The expression vector is pPWW50A and / or pMV261, and the host cell is Streptomyces coelicolor M1146 and / or Mycobacterium smegmatis MC 2 155.

3. The biosynthetic method according to claim 2, wherein, The P450 dimerization enzyme or mutants thereof is selected from any one of the following: A1) AcolP450 dimerization enzyme with an amino acid sequence as shown in SEQ ID NO: 1, or a protein having more than 90% sequence identity with the amino acid sequence as shown in SEQ ID NO: 1 and having the same function; A2) AchaP450 dimerization enzyme with an amino acid sequence as shown in SEQ ID NO: 2, or a protein having more than 90% sequence identity with the amino acid sequence as shown in SEQ ID NO: 2 and having the same function; A3) StsuP450 dimerization enzyme with an amino acid sequence as shown in SEQ ID NO: 3, or a protein having more than 90% sequence identity with the amino acid sequence as shown in SEQ ID NO: 3 and having the same function; A4) StnoP450 dimerization enzyme with an amino acid sequence as shown in SEQ ID NO: 4, or a protein having more than 90% sequence identity with the amino acid sequence as shown in SEQ ID NO: 4 and having the same function; A5) StsuP450-A398F mutant with an amino acid sequence as shown in SEQ ID NO: 5, or a protein having more than 90% sequence identity with the amino acid sequence as shown in SEQ ID NO: 5 and having the same function; A6) StsuP450-A398L mutant with an amino acid sequence as shown in SEQ ID NO: 6, or a protein having more than 90% sequence identity with the amino acid sequence as shown in SEQ ID NO: 6 and having the same function; A7) StsuP450-W184F mutant with an amino acid sequence as shown in SEQ ID NO: 7, or a protein having more than 90% sequence identity with the amino acid sequence as shown in SEQ ID NO: 7 and having the same function; A8) StsuP450-W184L mutant with an amino acid sequence as shown in SEQ ID NO: 8, or a protein having more than 90% sequence identity with the amino acid sequence as shown in SEQ ID NO: 8 and having the same function; A9) a StsuP450-W184A mutant having an amino acid sequence as shown in SEQ ID NO: 9 or a protein having more than 90% sequence identity to the amino acid sequence as shown in SEQ ID NO: 9 and having the same function.

4. The biosynthetic method according to claim 3, characterized in that, The nucleic acid molecule encoding said P450 dimerization enzyme or a mutant thereof is selected from any one of: B1) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 10, or a nucleic acid molecule hybridizing with a nucleic acid molecule as defined under B1) and encoding an Acol P450 dimerization enzyme according to claim 3, or a nucleic acid molecule having more than 90% sequence identity to a nucleic acid molecule as defined under B1) and encoding an Acol P450 dimerization enzyme according to claim 3; B2) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 11, or a nucleic acid molecule hybridizing with a nucleic acid molecule as defined under B2) and encoding an Acha P450 dimerization enzyme according to claim 3, or a nucleic acid molecule having more than 90% sequence identity to a nucleic acid molecule as defined under B2) and encoding an Acha P450 dimerization enzyme according to claim 3; B3) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 12, or a nucleic acid molecule hybridizing with a nucleic acid molecule as defined under B3) and encoding a Stsu P450 dimerization enzyme according to claim 3, or a nucleic acid molecule having more than 90% sequence identity to a nucleic acid molecule as defined under B3) and encoding a Stsu P450 dimerization enzyme according to claim 3; B4) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 13, or a nucleic acid molecule hybridizing with a nucleic acid molecule as defined under B4) and encoding a Stno P450 dimerization enzyme according to claim 3, or a nucleic acid molecule having more than 90% sequence identity to a nucleic acid molecule as defined under B4) and encoding a Stno P450 dimerization enzyme according to claim 3; B5) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 14, or a nucleic acid molecule hybridizing with a nucleic acid molecule as defined under B5) and encoding a Stsu P450-A398F mutant according to claim 3, or a nucleic acid molecule having more than 90% sequence identity to a nucleic acid molecule as defined under B5) and encoding a Stsu P450-A398F mutant according to claim 3; B6) a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID NO: 15, or a nucleic acid molecule hybridizing with a nucleic acid molecule as defined under B6) and encoding a Stsu P450-A398L mutant according to claim 3, or a nucleic acid molecule having more than 90% sequence identity to a nucleic acid molecule as defined under B6) and encoding a Stsu P450-A398L mutant according to claim 3; B7) a nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO: 16, or a nucleic acid molecule that hybridizes to the nucleic acid molecule defined in B7) and encodes the StsuP450-W184F mutant of claim 3, or a nucleic acid molecule that has more than 90% sequence identity to the nucleic acid molecule defined in B7) and encodes the StsuP450-W184F mutant of claim 3; B8) a nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO: 17, or a nucleic acid molecule that hybridizes to the nucleic acid molecule defined in B8) and encodes the StsuP450-W184L mutant of claim 3, or a nucleic acid molecule that has more than 90% sequence identity to the nucleic acid molecule defined in B8) and encodes the StsuP450-W184L mutant of claim 3; B9) a nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO: 18, or a nucleic acid molecule that hybridizes to the nucleic acid molecule defined in B9) and encodes the StsuP450-W184A mutant of claim 3, or a nucleic acid molecule that has more than 90% sequence identity to the nucleic acid molecule defined in B9) and encodes the StsuP450-W184A mutant of claim 3.

5. The biosynthetic method according to claim 1, characterized in that, In step S1, the inducing expression step specifically comprises: when the culture reaches OD 600 When the value is 0.8-1.0, acrylamide with a mass concentration of 0.1-0.5%, ALA with a mass concentration of 0.05-0.2%, and Fe(NH4)2(SO4)2 with a mass concentration of 0.05-0.2% are added, and the induction is carried out at a temperature of 25-30°C and a rotation speed of 200-240 rpm for 12-16 h.

6. The biosynthetic method of claim 1, wherein, In step S2, the concentration of the tryptophan-containing cyclic dipeptide substrate is 80-120 μM, and the tryptophan-containing cyclic dipeptide substrate is at least one selected from cWW, cWF, cWY, cWL, cWA, cWM, cWV, cWI, cWP, cWS, cWG, 5,5'-difluoro-cWW, 6,6'-difluoro-cWW, 6,6'-dichloro-cWW, 6,6'-dibromo-cWW.

7. The biosynthetic method of claim 1, wherein, In step S2, the catalytic reaction specifically comprises: under the condition that the temperature is 25-30℃ and the rotation speed is 700-900 rpm, reacting for 2-4 d.

8. A diketopiperazine heterodimer synthesized by the biosynthetic method of any one of claims 1-7.

9. The diketopiperazine heterodimer of claim 8, wherein, The diketopiperazine heterodimer comprises the structure shown in the following formulas 1-25: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 10. Use of the diketopiperazine heterodimer of claim 8 or 9 in the preparation of an anti-inflammatory, anticancer, antimalarial, or antiviral drug.

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