Recombinant zearalane-producing strain, construction method and application thereof

Abstract

The present application relates to the field of synthetic biology and genetic engineering technology, and particularly relates to a recombinant strain for producing Zearalane, wherein the recombinant strain takes a four-deficient Aspergillus oryzae as a host cell, and a polyketide synthase gene 111A and a polyketide synthase gene 111B are heterogenously co-expressed in the host cell; the DNA sequence of the polyketide synthase gene 111A is shown as SEQ ID NO:1, and the DNA sequence of the polyketide synthase gene 111B is shown as SEQ ID NO:2; the polyketide synthase gene 111A and the polyketide synthase gene 111B are restructured in the host cell through an independently constructed expression vector. In the present application, two key PKS coding genes derived from a wild-type fungus are restructured into a four-deficient Aspergillus oryzae host, stable co-expression of a double-plasmid system and coordinated supply of precursors are realized, and a hidden biosynthesis gene cluster in a natural state is activated.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and genetic engineering, specifically to a recombinant strain that produces Zearalane, its construction method, and its applications. Background Technology

[0002] Zearalane (a class of zearalenone compounds) is a natural product of resorcylic acid lactones (RAL) biosynthesized by fungi via the polyketide pathway. Its molecular formula is C2. 18 H 26 O4, with a molecular weight of 306.40, is characterized by a core chemical structure containing a macrocyclic lactone framework fused with a resorcinol aromatic unit. Chemically, it belongs to the saturated derivatives of the zearalenone family. These compounds exhibit estrogen-like activity, specifically binding to estrogen receptors in mammals, and are typical mycoestrogen-derived natural active molecules. Zearalenone and its derivatives have shown significant application value in animal growth regulation, endocrine regulation, and pharmacological activity research. For example, its homologue, zearalenol, has been used as a growth promoter in livestock farming for cattle, sheep, and other economically important animals. Furthermore, zearalenone compounds have shown potential applications in research models related to endocrine regulation, immune system regulation, anti-inflammatory mechanisms, and cell signal transduction regulation.

[0003] In nature, Zearalane compounds are mainly produced by Fusarium sowii (…). Fusarium graminearum Fusarium pinkii ( ) Fusarium roseum ) and some wild-type *Metacarpa* species ( Diaporthe Plant endophytic fungi produce this macrocyclic lactone backbone via a secondary metabolic pathway mediated by polyketide synthases (PKS). From a biosynthetic perspective, the assembly process of this backbone is quite complex, typically involving the synergistic action of highly reduced polyketide synthases (hrPKS) and non-reduced polyketide synthases (nrPKS) in both time and space. In this process, hrPKS is responsible for assembling and alternately reducing the polymer chain to form a partially saturated polyketide chain tip. Subsequently, nrPKS takes over the chain, continuing non-reducing polyketide chain extension, aromatic cyclization, and lactone formation. Finally, it undergoes the action of a later-stage reducing enzyme to form the target saturated backbone.

[0004] However, in wild-type strains from natural sources, the gene cluster controlling the biosynthesis of this compound (the 111 gene cluster) is affected by the strain's own epigenetic regulation or transcriptional repression. Under conventional laboratory culture or fermentation conditions, the 111 gene cluster is often in a silent or cryptic state. This makes it difficult to detect or isolate the target Zearalane compounds from the secondary metabolites of wild-type strains. Traditional methods of obtaining these substances through natural harvesting and wild-type fermentation are limited, resulting in a natural resource bottleneck for subsequent pharmacological evaluation, systems toxicology studies, and industrial development.

[0005] Although these compounds can be prepared through all-chemical synthesis, their structures contain multi-center chiral structures, macrocyclic lactone rings, and resorcinol structural units that are sensitive to reaction conditions. This results in long synthetic routes, cumbersome steps, low overall yields, poor stereoselectivity, and the use of environmentally unfriendly reagents. Therefore, assembling and reconstructing the hrPKS and nrPKS synergistic biosynthetic pathway in engineered heterologous hosts using synthetic biology techniques, thereby improving their biosynthetic efficiency, has significant scientific research and industrialization value.

[0006] Aspergillus oryzae ( Aspergillus oryzae As a filamentous fungus industrial chassis cell, *Aspergillus oryzae* is recognized as a Generally Recognized As Safe (GRAS) strain by the U.S. Food and Drug Administration (FDA). *Aspergillus oryzae* possesses a clear genetic background, strong post-translational modification capabilities, and eukaryotic intron splicing mechanisms, making it suitable for expressing multifunctional enzymes (such as PKS) derived from fungal secondary metabolic pathways. However, existing heterologous expression systems often face challenges such as plasmid instability, low efficiency of co-expression of multiple exogenous genes, and insufficient precursor supply when dealing with the co-expression of large-fragment dual PKS genes. Summary of the Invention

[0007] The present invention aims to provide a recombinant strain that produces Zearalane by reconstructing two key PKS encoding genes derived from wild-type fungi into a four-deficient Aspergillus oryzae chassis host, thereby achieving stable co-expression of the dual plasmid system and synergistic precursor supply, and activating the naturally silent cryptic biosynthetic gene cluster.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: Objective 1 of this invention: A recombinant strain producing Zearalane is provided; the recombinant strain uses Aspergillus tetradeficient as a chassis host cell, and heterologously co-expresses polyketide synthase genes 111A and 111B in the chassis host cell. The DNA sequence of polyketide synthase gene 111A is shown in SEQ ID NO:1, and the DNA sequence of polyketide synthase gene 111B is shown in SEQ ID No:2; polyketide synthase genes 111A and 111B are synergistically reconstructed in the chassis host cell through independently constructed expression vectors.

[0009] Furthermore, the polyketide synthase gene 111A and polyketide synthase gene 111B are derived from the original fungal strain. Diaporthe kyushuensis The 111 gene cluster of ZMU-48-1.

[0010] Furthermore, the chassis host cell is the filamentous fungus *Aspergillus tetradeficientis*. NSAR1 This strain has a genotype that is nitrate reductase deficient (niaD). - ), ATP sulfate deficiency (sC - Adenine synthesis pathway defect (adeA) - Ornithine carbamoyltransferase deficiency (argB) - (4) Quadruple nutritional deficiency markers.

[0011] The second objective of this invention is: A method for constructing the recombinant strain is provided, comprising the following steps: (1) Amplification of target genes: Using the genomic DNA or cDNA of the four-deficient Aspergillus oryzae strain as a template, the polyketide synthase gene 111A fragment and the polyketide synthase gene 111B fragment were amplified by specific primer pairs. (2) Construction of recombinant expression plasmids: The polyketide synthase gene 111A fragment was cloned and ligated into the pUSA vector to construct the recombinant expression plasmid pUSA-111A with the sC defective complementation marker; the polyketide synthase gene 111B fragment was cloned and ligated into the pTaex3 vector to construct the recombinant expression plasmid pTaex3-111B with the argB defective complementation marker; (3) Co-transformation and screening of host protoplasts: preparation of four-deficient Aspergillus oryzae Aspergillus oryzaeNSAR1 The host cell protoplasts; under PEG-mediated transformation, the recombinant expression plasmids pUSA-111A and pTaex3-111B were co-transformed into the protoplasts, and double screening was performed on complementary auxotrophic selection medium to select stable genetically positive double transformants, thus obtaining the recombinant strain.

[0012] Further, in step (2), the polyketide synthase gene 111A is transcribed and expressed in the recombinant expression plasmid pUSA-111A by the promoter TAmyA; the polyketide synthase gene 111B is expressed in the expression plasmid pTaex3-111B by the promoter TAmyA. amyB Drives transcriptional expression.

[0013] The third objective of this invention: The claim seeks protection for the use of the recombinant strain in the preparation or fermentation production of the compound Zearalane.

[0014] Furthermore, the application involves using the recombinant strain and / or its crude fermentation extract to prepare animal growth regulators, endocrine regulators, immunomodulators, anti-inflammatory drugs, or reagents for cell signal transduction research.

[0015] The fourth objective of this invention is: A method for preparing the compound Zearalane by fermentation using the recombinant strain is provided, comprising the following steps: inoculating the recombinant strain into a fermentation medium containing an exogenous carbon source inducer for large-scale fermentation culture; after fermentation, collecting the fermentation broth and cell products, extracting them with an organic solvent to obtain a crude extract; and performing chromatographic separation and purification on the crude extract to obtain the pure compound Zearalane.

[0016] Furthermore, the fermentation medium is DPY liquid medium, and the exogenous carbon source inducer is maltose.

[0017] Furthermore, the organic solvent is of low or medium polarity.

[0018] Compared with the prior art, the beneficial effects of the present invention are: (1) Activation of silent gene clusters to significantly increase the yield of target products: This invention identifies and clones the core hrPKS and nrPKS of the 111 gene cluster in the original strain and places them under the transcriptional system driven by the Aspergillus oryzae chassis, thereby relieving the transcriptional repression in the native state. In the fermentation system, this hidden biosynthetic pathway is activated, increasing the yield of Zearalane in the fermentation broth from undetectable in the wild type to 3.88 g / L, effectively solving the problem of limited natural isolation and acquisition of this compound.

[0019] (2) High precursor supply fit and reduced interference from background metabolic impurities: The endogenous metabolic flux of Aspergillus oryzae chassis cells is vigorous. The dual plasmid expression vector system (pUSA-111A + pTaex3-111B) reconstructed in this invention can adapt well to the host's precursor flux, enabling the synergistic reconstruction of two large gene fragments. Due to the low amount of endogenous secondary metabolic impurities in the Aspergillus oryzae fermentation system, the fermentation baseline is stable, effectively reducing the difficulty of subsequent separation and purification processes.

[0020] (3) Good industrial application foundation: The four-defect Aspergillus oryzae host adopted belongs to the industrial safety certification (GRAS) chassis. The fermentation process is mild. Compared with the complex chemical synthesis of long routes, this biosynthesis scheme avoids the excessive use of heavy metal catalysts and organic reagents, which is in line with the concept of green pharmaceutical and sustainable development. Attached Figure Description

[0021] Figure 1 The original strain in this invention Diaporthe kyushuensis A schematic diagram of the physical map region of the 111 biosynthetic gene cluster (Cluster111) in ZMU-48-1; Figure 2 The topological structure diagram of the recombinant expression plasmid pUSA-111A constructed in this invention is shown. Figure 3 The topological structure diagram of the recombinant expression plasmid pTaex3-111B constructed in this invention; Figure 4 This is a high-performance liquid chromatography (HPLC) chromatogram of the fermentation extract of the recombinant strain of the present invention; wherein, cluster 111.1 represents the newly generated Zearalane product peak in the recombinant strain; blank AO represents... AspergillusoryzaeNSAR1 The Zearalane product peak produced by a blank host.

[0022] Figure 5 The figure shows the quantitative analysis results of the target compound Zearalane obtained by fermentation purification; Figure 6 For the target compound Zearalane 1 H NMR spectrum; Figure 7 For the target compound Zearalane 13 C NMR spectrum; Figure 8 For the target compound Zearalane 1 H– 1 H COSY spectrum; Figure 9 The HSQC spectrum of the target compound Zearalane; Figure 10 The HMBC spectrum of the target compound Zearalane; Figure 11 The image shows the characteristic UV absorption of the target compound, Zearalane. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited to the following embodiments. For those skilled in the art, any equivalent substitutions or variations made without departing from the concept of the present invention should fall within the protection scope of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are performed in accordance with conventional standards such as *Molecular Cloning: A Laboratory Manual*; unless otherwise specified, the reagents used are commercially available.

[0024] The specific formula for DPY liquid culture medium is as follows: 20 g dextrin, 10 g polypeptone, 5 g yeast extract, 0.5 g magnesium sulfate heptahydrate (MgSO47H2O), 5 g potassium dihydrogen phosphate (KH2PO4), and water is added to a final volume of 1000 mL.

[0025] Example 1: Cloning of diketone synthase genes 111A and 111B and assembly of recombinant expression vector (1) Acquisition of target gene fragments: Extraction of freshly cultured plant endophytic fungi Diaporthe kyushuensis High molecular weight genomic DNA from ZMU-48-1 (accession number GDMCC No: 66577). Amplification primers containing recombinant arms were designed based on gene boundaries (see attached). Figure 1 The primer sequences are as follows: Primer 111A-F: 5'-AGCTACTACAGATCCCCGGTACCTGCCGAGAGTAACTAAGTCAGT-3'; Primer 111A-R: 5'-AAGCTGAATTCGAGCTCGGTACCAAAGGAATCATGCAACGATTGAG-3'; Primer 111B-F: 5'-CTCCGAATTCGAGCTCGGTACCTTCATGGCCACAACAATGCC-3'; Primer 111B-R: 5'- GAGCTACTACAGATCCCCGGTACCGATCAAGCTTGACTAGACG-3'.

[0026] PCR amplification was performed using a high-fidelity DNA polymerase (such as Phanta Flash Super-Fidelity DNA Polymerase). The PCR reaction conditions were set as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, annealing (temperature set to Tm+5℃ based on primer Tm value) for 5 s, extension at 72℃ for 40 s, for a total of 30 cycles; and a final extension at 72℃ for 1 min. After the reaction, the PCR products were detected by agarose gel electrophoresis, and the amplified and recovered fragments of the partially reduced polyketide synthase (hrPKS) gene 111A (DNA sequence SEQ ID NO:1) and the non-reduced polyketide synthase (nrPKS) gene 111B (DNA sequence SEQ ID NO:2) with a length of 6819 bp were obtained.

[0027] (2) Cloning and assembly of recombinant expression plasmids: Assembly of pUSA-111A: The pUSA vector was linearized using the restriction endonuclease KpnI. Using homologous recombination cloning technology, the 6819 bp 111A fragment obtained above was recombinated and ligated into the linearized vector at 50℃ for 30 minutes. The recombinant product was transformed into... E. coli In DH5α competent cells, the recombinant expression plasmid pUSA-111A carrying the sC defective complementary marker was obtained through PCR verification and sequencing identification. Figure 2 ).

[0028] Assembly of pTaex3-111B: The restriction endonuclease KpnI was used to synthesize pTaex3-111B with the promoter... amyB The pTaex3 vector was linearized and cut. The 7692 bp gene fragment 111B was directionally cloned into the vector using homologous recombination. amyB Downstream of the promoter, after transformation and sequencing verification, the recombinant expression plasmid pTaex3-111B with the argB defective complementary marker was constructed (see attached image). Figure 3 ).

[0029] Example 2: Preparation of four-deficient Aspergillus oryzae protoplasts and co-transformation with two plasmids (1) Pre-culture and preparation of mycelium: Prepare exogenous expression plasmids (pUSA-111A and pTaex3-111B), ensuring the plasmid concentration is >1 μg / μL for later use. The chassis receptor cell host strain... Aspergillus oryzaeNSAR1Perform inoculum resuscitation. Inoculate the inoculum into DPY liquid medium and incubate at 30°C and 150 rpm with shaking for 1-2 days. Under aseptic conditions, use a sterile scalpel to mince the mycelium, then continue suspension culture under the same conditions to prepare a seed culture. Transfer the seed culture to 500 mL Erlenmeyer flasks (each flask contains 200 mL of fresh DPY liquid medium) and incubate at 30°C and 120-150 rpm with shaking for 3-5 days. Once the mycelium has fully colonized the flask, aseptically filter and collect the mycelium.

[0030] (2) Preparation and decanting of the composite lysis buffer: 0.7 g of ammonium sulfate ((NH4)2SO4), 0.1 g of snail enzyme, and 0.1 g of yatalase were added sequentially to a 15 mL centrifuge tube to obtain the composite lysis buffer. Buffer TF0 was added to the tube until the total volume reached 10 mL. The tube was slowly inverted to dissolve the enzyme, avoiding vigorous shaking that could generate bubbles that would affect enzyme activity. After the enzyme components were dissolved and clarified, they were filtered through a 0.22 μm microporous membrane for sterilization to obtain the composite lysis buffer. The previously collected free bacterial cake was dispersed and poured into the composite lysis buffer. The enzymatic hydrolysis system was placed in a constant temperature shaker at 30℃ and 90-105 rpm for approximately 3 hours for the enzymatic hydrolysis reaction.

[0031] (3) Filtration and Purification Washing: The enzymatically digested reaction solution was first filtered through a non-woven fabric filter bag, and the filtrate was collected into a 50 mL centrifuge tube; then, a second-stage filtration was performed using a syringe filter with a cotton liner. An equal volume (10 mL) of washing buffer TF2 was added to the collected protoplast filtrate, and the mixture was mixed by inverting. The mixture was centrifuged at 1500-1600 rpm at 4°C for 10 minutes, and the supernatant was discarded. 5-10 mL of TF2 buffer was added to the precipitate at the bottom of the tube, and the tube was gently tapped to resuspend the precipitate evenly, avoiding vigorous pipetting. The mixture was centrifuged again at 1500 rpm for 10 minutes, and the supernatant was discarded. The mixture was washed twice with TF2 buffer as described above. A drop of the purified suspension was aspirated onto a hemocytometer for microscopic counting, and the cells were diluted or concentrated with TF2 buffer to ensure a cell concentration of 120-140 protoplasts per large square.

[0032] (4) PEG-mediated co-transformation: Transfer 200 μL of properly prepared protoplast suspension to a sterile centrifuge tube. Add 10 μL of recombinant expression plasmid pUSA-111A vector DNA at a concentration of not less than 1000 ng / μL; if the plasmid concentration is approximately 700 ng / μL, increase the volume to 14 μL (add recombinant expression plasmid pTaex3-111B at the same mass ratio). Mix well after addition, incubate on ice for 30 minutes, then transfer to room temperature (25℃) for static incubation for 20 minutes. Gently pipette to mix, and continue incubation at room temperature for 20 minutes. Then add PEG fusion buffer TF3 to the transformation system. Centrifuge at 1500 rpm for 5 minutes at room temperature, remove most of the supernatant, leaving approximately 200 μL of residual suspension at the bottom of the tube. Add washing buffer TF2 to bring the volume to 400 μL, and gently mix to obtain the transformant suspension.

[0033] (5) Spreading and screening: Take 200 μL of the prepared transformant suspension and spread it in the center of the lower solid medium plate of M+ADE (M refers to Aspergillus oryzae minimal medium; +ADE indicates the addition of adenine at a concentration of 0.1 g / L). Add the upper medium to solidify. Place the plate in a 30℃ incubator and invert it for 3-5 days until single colonies of transformants grow on the surface. Pick single colonies and passage them for at least 3 generations in the appropriate screening medium to integrate the exogenous gene fragment into the chromosome set, thereby obtaining a recombinant Aspergillus oryzae strain that can be stably inherited.

[0034] Basic culture medium for Aspergillus oryzae: To prepare 1L of medium, you need 20 g glucose, 2 g ammonium chloride (NH4Cl), 1 g potassium dihydrogen phosphate (KH2PO4), 0.5 g potassium chloride (KCl), 0.5 g sodium chloride (NaCl), 0.02 g ferrous sulfate heptahydrate (FeSO4·7H2O), 0.5 g magnesium sulfate heptahydrate (MgSO4·7H2O), 218.69 g sorbitol, and 18 g agar.

[0035] Buffer TF0: Primarily used to provide a slightly acidic buffer environment to maintain optimal activity of the wall-lysing complex enzyme. 50 mM maleic acid, with the pH adjusted to 5.5 using NaOH.

[0036] Buffer TF2: Its main functions are to wash away free impurities and complex enzymes, maintain the hypertonic environment of protoplasts, and provide calcium ions to stabilize the fragile protoplast cell membrane. 1.2 M sorbitol, 50 mM CaCl2, 35 mM NaCl, 10 mM Tris-HCl (pH 7.5).

[0037] Buffer TF3: Its main function is to utilize high concentrations of polyethylene glycol to alter protoplast membrane permeability, efficiently mediating the transmembrane entry of exogenous recombinant plasmid DNA into the cell. 60% ( w / v ) PEG 4000, 50 mM CaCl2, 10 mM Tris-HCl (pH7.5).

[0038] Example 3: Micro-culture, extraction and quantitative analysis of recombinant engineered strains (1) Small-scale fermentation culture: The target recombinant Aspergillus oryzae strain, verified by subculturing, was inoculated into the seed culture for activation, and then transferred to a 500 mL Erlenmeyer flask containing fermentation induction medium at an inoculation rate of 8%-10%, with a fermentation volume of 200 mL. The fermentation induction medium used was DPY liquid medium. Maltose was used as an exogenous inducer to activate the fermentation induction medium. amyB Promoter. Fermented continuously for 7 days at 30℃ and 150 rpm. As a parallel control, a wild-type fungal strain was used. Diaporthe kyushuensis ZMU-48-1 was fermented in 200 mL under the same conditions.

[0039] (2) Extraction of the target product: After fermentation, the fermentation broth was collected. An equal volume of anhydrous ethanol was added to ultrasonically break down and extract the Aspergillus oryzae mycelium, and the process was repeated 3 times. The extracts were filtered and combined, and the ethanol was recovered by vacuum concentration to obtain an aqueous suspension. The suspension was transferred to a separatory funnel and an equal volume of ethyl acetate was added for extraction. The extraction was repeated 3 times, and the ethyl acetate organic phases were combined and concentrated to dryness under vacuum at 50°C to obtain the crude fermentation extract.

[0040] (3) Sample dilution and chromatographic quantitative detection: Dissolve the crude extract of 200 mL fermentation broth in chromatographic grade methanol and bring the volume up to 30 mL. Use a pipette to measure 100 μL of the sample stock solution and add it to a centrifuge tube containing 900 μL of methanol to dilute evenly and obtain a detection loading solution with a total volume of 1 mL (diluted 10 times). The test solution was filtered through a 0.22 μm syringe filter and analyzed using a C18 reversed-phase column (4.6 mm × 250 mm, 5 μm). Mobile phase A was chromatographic grade acetonitrile, and mobile phase B was ultrapure water (or 0.1% formic acid aqueous solution). The flow rate was controlled at 1.0 mL / min, and the injection volume was 10 μL. The gradient elution program was set as follows: the proportion of mobile phase A increased linearly from 10% to 50% within 0–10 min; the proportion of mobile phase A increased linearly from 50% to 100% within 10–20 min; the proportion of mobile phase A was maintained at 100% within 20–24 min; and the proportion of mobile phase A was reduced to 10% and equilibrated for 6 min within 24–30 min. Compared with the control group blank AO, a significant Zearalane metabolic peak was observed (see attached image). Figure 4 A standard regression curve for external standard quantification with six concentration gradients was constructed using Zearalane standards. The equation is: Y = 2730.1736X +32.4695 (Y is the peak area, and X is the concentration).

[0041] (4) Titer calculation and result analysis: Substituting the measured peak area Y = 7102.171 of the crude extract from the fermentation of the target recombinant Aspergillus oryzae engineered strain into the standard curve formula Y = 2730.1736X + 32.4695, the concentration of Zearalane in 1 mL of the detection loading solution was calculated as follows:

[0042] Since the working solution for this test was prepared by uniformly diluting 30 mL of extract stock solution by 10 times, the concentration of Zearalane in the 30 mL extract stock solution is:

[0043] The total mass of Zearalane contained in this 30 mL system ( M total )for:

[0044] Given that the total mass of the target product ultimately originates from an initial fermentation broth system of 200 mL (i.e., 0.2 L), the final fermentation production titer (yield) of this recombinant Aspergillus oryzae engineered strain is... Y The calculation is as follows:

[0045] In a 200 mL fermentation system, the recombinant Aspergillus oryzae engineered strain achieved a yield of 3.88 g / L of the target product Zearalane (111-1 macrolide (DAD1A)). Figure 5 Quantitative results from comparisons show that, through heterologous reconstruction of the 111A and 111B diketone synthase genes, this invention activates the low-expression or silenced biosynthetic gene clusters in the original strain of Aspergillus oryzae in the Aspergillus oryzae system, thereby increasing the yield of the target macrocyclic lactone compound.

[0046] Example 4: Structural confirmation of the target product Zearalane 1. Identify the structure of the obtained compound using the following spectral methods (see attached diagram). Figure 6-10 ): (1) 1D NMR: 1 H NMR, 13 C NMR, DEPT; (2) 2D NMR: 1 H– 1 H COSY, HSQC, HMBC.

[0047] The compound is a needle-like crystal with the molecular formula C0. 18 H 26 O4, in methanol solution, has the highest UV absorption. max =218.0, 262.9, 302.1 nm (attached) Figure 11 ). 1 H NMR (600 MHz, CDCl3) δ H 6.27 (d, J = 2.6Hz, H-4), 6.22 (d, J = 2.6Hz, H-6), 5.20 (dq, J = 12.0, 6.2 Hz, H-17), 3.26 (td, J =12.5, 4.8 Hz, H-8a), 2.43(td, J = 12.5, 4.8 Hz, H-8b), 1.89 – 1.21 (m, H-9 / 10 / 11 / 12 / 13 / 14); 13 C NMR (150 MHz, CDCl3) δ C171.9 (s, C-1), 165.8 (s, C-3), 160.4 (s, C-5), 149.3 (s, C-7), 110.7 (d, C-6) , 105.5 (s, C-2) , 101.6 (d, C-4) ,73.8 (t, C-17) , 37.4 (t, C-8) , 34.9 (t, C-16) , 31.4 (t, C-9) , 27.0 (t, C-11), 26.9 (t, C-12) , 26.8 (t, C-10) , 22.8 (t, C-15) ,22.7 (t, C-13) , 22.6(t, C-14) , 21.5 (q, C-18). Therefore, it was identified as Zearalane.

[0048] The results confirmed that synergistic catalysis was achieved using the 111A and 111B genes within the recombinant chassis system, resulting in the synthesis of the target product Zearalane. The chemical molecular structure of Zearalane is as follows: .

[0049] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A recombinant strain producing Zearalane, characterized in that: The recombinant strain uses Aspergillus tetradeficient as the chassis host cell, and heterologously co-expresses polyketide synthase genes 111A and 111B within the chassis host cell. The DNA sequence of polyketide synthase gene 111A is shown in SEQ ID NO:1, and the DNA sequence of polyketide synthase gene 111B is shown in SEQ ID No:

2. Polyketide synthase genes 111A and 111B are synergistically reconstructed within the chassis host cell through independently constructed expression vectors.

2. The recombinant strain producing Zearalane according to claim 1, characterized in that: The polyketide synthase gene 111A and polyketide synthase gene 111B are derived from the original fungal strain. Diaporthe kyushuensis The 111 gene cluster of ZMU-48-1.

3. The recombinant strain producing Zearalane according to claim 2, characterized in that: The chassis host cell is the filamentous fungus *Aspergillus oryzae*, a tetra-deficient fungus. NSAR1 The strain has a genotype with four auxotrophic markers: nitrate reductase deficiency, ATP sulfate deficiency, adenine synthesis pathway deficiency, and ornithine carbamoyltransferase deficiency.

4. The method for constructing the recombinant strain according to any one of claims 1 to 3, characterized in that: Includes the following steps: (1) Amplification of target genes: Using the genomic DNA or cDNA of the four-deficient Aspergillus oryzae strain as a template, the polyketide synthase gene 111A fragment and the polyketide synthase gene 111B fragment were amplified by specific primer pairs. (2) Construction of recombinant expression plasmids: The polyketide synthase gene 111A fragment was cloned and ligated into the pUSA vector to construct the recombinant expression plasmid pUSA-111A with the sC defective complementation marker; the polyketide synthase gene 111B fragment was cloned and ligated into the pTaex3 vector to construct the recombinant expression plasmid pTaex3-111B with the argB defective complementation marker; (3) Co-transformation and screening of host protoplasts: preparation of four-deficient Aspergillus oryzae Aspergillus oryzaeNSAR1 The host cell protoplasts; under PEG-mediated transformation, the recombinant expression plasmids pUSA-111A and pTaex3-111B were co-transformed into the protoplasts, and double screening was performed on complementary auxotrophic selection medium to select stable genetically positive double transformants, thus obtaining the recombinant strain.

5. The method for constructing the recombinant strain according to claim 4, characterized in that: In step (2), the polyketide synthase gene 111A is transcribed and expressed in the recombinant expression plasmid pUSA-111A by the promoter TAmyA; the polyketide synthase gene 111B is expressed in the expression plasmid pTaex3-111B by the promoter TAmyA. amyB Drives transcriptional expression.

6. The use of the recombinant strain according to any one of claims 1 to 3 in the preparation of the compound Zearalane.

7. The application according to claim 6, characterized in that: The application involves using recombinant strains and / or their crude fermentation extracts to prepare animal growth regulators, endocrine-regulating drugs, immunomodulators, anti-inflammatory drugs, or reagents for cell signal transduction research.

8. A method for preparing the compound Zearalane by fermentation using the recombinant strain according to any one of claims 1 to 3, characterized in that: Includes the following steps: The recombinant strain was inoculated into a fermentation medium containing an exogenous carbon source inducer for large-scale fermentation culture; after fermentation, the fermentation broth and bacterial products were collected, and extracted with an organic solvent to obtain a crude extract; the crude extract was subjected to chromatographic separation and purification to obtain the pure compound Zearalane.

9. The method according to claim 8, characterized in that: The fermentation medium is DPY liquid medium, and the exogenous carbon source inducer is maltose.

10. The method according to claim 9, characterized in that: The organic solvent is of low or medium polarity.

Images