Aspergillus nidulans-derived fatty acid beta-oxidation pathway coding gene and application thereof

Abstract

The invention discloses a fatty acid beta-oxidation pathway coding gene derived from aspergillus nidulans and application of the fatty acid beta-oxidation pathway coding gene. The coding gene is selected from the following groups: (a) an Aspergillus 0G048390 gene with a nucleotide sequence as shown in SEQ ID NO: 1; (b) an Aspergillus 0G044330 gene of which the nucleotide sequence is as shown in SEQ ID NO: 3; and / or (c) an Aspergillus 0G004350 gene of which the nucleotide sequence is as shown in SEQ ID NO: 5. The engineering strain constructed by the invention has the advantages that the yield of echinocandin B is obviously increased; in shake flask fermentation, the yield of the overexpressed strain is improved by 33.55%, 25.14% and 25.62% compared with that of the original strain, important technical support is provided for industrial efficient production of echinocandin B, and the Echinocandin B strain has remarkable economic value and application prospect.

Description

Technical Field

[0001] This application belongs to the field of genetic engineering and relates to the fatty acid β-oxidation pathway encoding gene derived from Aspergillus nidulans and its application, especially its application in the industrial-scale high-efficiency fermentation production of echinocandin B. Background Technology

[0002] Invasive fungal infections have become a major challenge in global public health. Existing antifungal drugs suffer from problems such as strong toxicity, frequent drug resistance, and limited efficacy. Echinocandin B (ECB), as a novel antifungal drug precursor, works by inhibiting fungal cell wall β-1,3-glucan synthase. It has advantages such as broad-spectrum antibacterial activity and low toxicity, and is a core raw material for the synthesis of clinical drugs such as anifenfenadine, with urgent market demand.

[0003] Currently, ECB is mainly produced through fermentation by Aspergillus nidulans, but the yield of natural strains is low (the yield of original strains in shake-flask fermentation is usually less than 1500 mg / L), which is difficult to meet the needs of industrial production. The biosynthesis of ECB belongs to the non-ribosomal peptide synthesis pathway, and the construction of its core structure requires acetyl-CoA as a key precursor. The fatty acid β-oxidation pathway is one of the important supply pathways of intracellular acetyl-CoA in Aspergillus nidulans. This pathway degrades fatty acids into acetyl-CoA through stepwise catalysis by key enzymes such as acyl-CoA oxidase (FOX1), 3-hydroxyacyl-CoA dehydrogenase (FOX2), and thiolase (POT1). Each step of the catalytic reaction is a potential regulatory node for the pathway flux, directly affecting the precursor supply efficiency of ECB synthesis.

[0004] In existing technologies, strategies for increasing ECB yield mainly focus on optimizing the electron transport system (such as overexpressing cytochrome P450 reductase genes) and regulating fermentation conditions. However, there are no reports on the discovery, identification, and individual overexpression of key enzyme genes in the fatty acid β-oxidation pathway. Therefore, screening core functional genes in the Aspergillus nidulans fatty acid β-oxidation pathway and enhancing the efficiency of a single catalytic step through individual overexpression, thereby increasing the acetyl-CoA supply flux, is an effective strategy for targeted improvement of ECB yield and is of great significance for promoting its industrial production.

[0005] Therefore, there is an urgent need to develop an engineered bacterium that produces echinocandin B with high ECB yield, simple strain construction, and strong genetic stability. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a gene encoding the fatty acid β-oxidation pathway derived from Aspergillus nidulans and its application, in order to solve the technical problem of low ECB yield in existing Aspergillus nidulans strains and achieve efficient biosynthesis of ECB.

[0007] To achieve the above and other related objectives, the following technical solution is adopted:

[0008] The first object of the present invention is to provide a gene encoding the fatty acid β-oxidation pathway derived from Aspergillus nidulans, said gene being selected from the group consisting of: (a) the Aspergillus0G048390 gene with the nucleotide sequence as shown in SEQ ID NO:1; (b) the Aspergillus0G044330 gene with the nucleotide sequence as shown in SEQ ID NO:3; and / or (c) the Aspergillus0G004350 gene with the nucleotide sequence as shown in SEQ ID NO:5.

[0009] Due to the specific nature of nucleotide sequences, any variant of the polynucleotide shown in SEQ ID NO.1, SEQ ID NO.3, or SEQ ID NO.5, provided it shares more than 90% homology with the original polynucleotide, falls within the scope of protection of this invention. The term "polynucleotide mutant" refers to a polynucleotide sequence with one or more nucleotide alterations. This polynucleotide mutant can be a live or non-live allelic variant, including substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may involve the substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function encoding the amino acid.

[0010] A second object of the present invention is to provide a protein encoded by the fatty acid β-oxidation pathway encoding gene derived from Aspergillus nidulans, the amino acid sequence of which is shown in SEQ ID NO:2, SEQ ID NO:4 and / or SEQ ID NO:6.

[0011] As a preferred embodiment of this application, SEQ ID NO:2 corresponds to the protein encoded by the Aspergillus0G048390 gene with the nucleotide sequence shown in SEQ ID NO:1. This protein is an acyl-CoA oxidase (FOX1) containing a conserved FOX1 domain, which is responsible for catalyzing the first step of fatty acid β-oxidation.

[0012] As a preferred embodiment of this application, SEQ ID NO:4 corresponds to the protein encoded by the Aspergillus0G044330 gene with the nucleotide sequence shown in SEQ ID NO:3, which is a 3-hydroxyacyl-CoA dehydrogenase (FOX2) containing a FOX2 domain.

[0013] As a preferred embodiment of this application, SEQ ID NO:6 corresponds to the protein encoded by the Aspergillus0G004350 gene with the nucleotide sequence shown in SEQ ID NO:5, which is a thiolytic enzyme (POT1) containing the POT1 domain.

[0014] Due to the specificity of amino acid sequences, any fragment or variant of the polypeptide with the amino acid sequences shown in SEQ ID NO. 2, SEQ ID NO. 4, and SEQ ID NO. 6, such as its conserved variants, bioactive fragments, or derivatives, is within the scope of protection of this invention, provided that the fragment or variant of the polypeptide shares more than 95% homology with the aforementioned amino acid sequences. The alterations may include the deletion, insertion, or substitution of amino acids in the amino acid sequence; for conserved alterations of variants, the substituted amino acid has a similar structure or chemical properties to the original amino acid, such as replacing isoleucine with leucine; variants may also have non-conserved alterations, such as replacing glycine with tryptophan.

[0015] A third objective of this invention is to provide a recombinant overexpression vector comprising a gene encoding the fatty acid β-oxidation pathway derived from Aspergillus nidulans, and an operable promoter and terminator.

[0016] As a preferred embodiment of this application, the recombinant overexpression vector is obtained by inserting a fatty acid β-oxidation pathway coding gene derived from Aspergillus nidulans as the base vector, and the coding gene is driven by the constitutive promoter gpdA and terminated by the terminator TtrpC.

[0017] A fourth objective of this invention is to provide a method for constructing a recombinant overexpression vector, comprising the following steps:

[0018] Three target genes were obtained by PCR amplification from the genome of Aspergillus nidulans ZJB16068: Aspergillus0G048390, Aspergillus0G044330, and Aspergillus0G004350.

[0019] By inserting a single target gene into the pDht-sk vector backbone linearized by single enzyme digestion (XbaⅠ) using a one-step cloning method, recombinant overexpression vectors pDht-sk-0G048390 containing the Aspergillus0G044330 gene, pDht-sk-0G044330 containing the Aspergillus0G004350 gene, and pDht-sk-0G004350 containing the Aspergillus0G004350 gene were constructed, which are the recombinant overexpression vectors.

[0020] The present invention also provides the application of the aforementioned encoding gene, the aforementioned protein, and the aforementioned recombinant overexpression vector in constructing genetically engineered bacteria that produce high levels of echinocandin B.

[0021] The fifth objective of this invention is to provide a genetically engineered bacterium containing the aforementioned coding gene or the aforementioned recombinant overexpression vector.

[0022] As a preferred embodiment of this application, the genetically engineered bacteria are Aspergillus nidulans, which produces echinocandin B, as the host bacteria. The recombinant overexpression vector is transformed into the host bacteria to obtain engineered strains that overexpress the Aspergillus0G048390 gene, the Aspergillus0G044330 gene, or the Aspergillus0G004350 gene.

[0023] As a preferred embodiment of this application, the host bacterium is Aspergillus nidulans ZJB16068.

[0024] The sixth objective of this invention is to provide a method for constructing the aforementioned genetically engineered bacteria, comprising the following steps:

[0025] (1) The coding genes for the fatty acid β-oxidation pathway were screened from the genome of Aspergillus nidulans ZJB16068; the coding genes are Aspergillus0G048390, Aspergillus0G044330 and / or Aspergillus0G004350.

[0026] (2) Using plasmid pDht-sk as the base vector, recombinant overexpression vectors containing Aspergillus0G048390, Aspergillus0G044330 and Aspergillus0G004350 genes were constructed respectively.

[0027] (3) The constructed recombinant overexpression vector was transformed into Aspergillus nidulans ZJB16068 using Agrobacterium AGL1-mediated transformation method to obtain three engineered strains that overexpressed the Aspergillus0G048390 gene, the Aspergillus0G044330 gene, and the Aspergillus0G004350 gene, which were named strain OE-0G048390, strain OE-0G044330, and strain OE-0G004350, respectively.

[0028] The seventh objective of this invention is to provide an application of the aforementioned encoding gene, the aforementioned protein, the aforementioned recombinant overexpression vector, the aforementioned genetically engineered bacteria, or the genetically engineered bacteria constructed by the method for constructing the aforementioned genetically engineered bacteria, in the fermentation production of echinocandin B.

[0029] The eighth objective of this invention is to provide a method for producing echinocandin B, wherein the genetically engineered bacteria are inoculated into a fermentation medium for fermentation culture to obtain echinocandin B.

[0030] As a preferred embodiment of this application, the fermentation conditions are: temperature 25℃, rotation speed 200r / min, and fermentation cycle 10h.

[0031] As a preferred embodiment of this application, the fermentation medium consists of 70% methyl oleate 90 g / L, Tween 80 10.98 g / L, sucrose 9.44 g / L, peanut oil 20 g / L, glycerol 10 g / L, soybean meal 40 g / L, sucrose 9.44 g / L, tryptone 8.6 g / L, threonine 3.1 g / L, ornithine hydrochloride 6.1 g / L, K2HPO4 6.1 g / L, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.2 g / L, FeSO4·7H2O 0.05 g / L, CaCl2 0.3 g / L, CuSO4·5H2O 0.6 g / L, with a natural pH.

[0032] This invention, through functional screening of genes related to the β-oxidation pathway in the Aspergillus nidulans genome, discovered that the three genes encode acyl-CoA oxidase (FOX1), 3-hydroxyacyl-CoA dehydrogenase (FOX2), and thiolase (POT1), respectively, constituting a core functional module for fatty acid β-oxidation. These genes can provide key precursors for echinocandin B synthesis by regulating intracellular acetyl-CoA metabolic flux. Overexpression of these three genes in echinocandin B-producing strains (Aspergillus nidulans and its mutants) significantly enhances the efficiency of the fatty acid β-oxidation pathway, increases acetyl-CoA supply, and thus directionally promotes the biosynthesis of echinocandin B.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] (1) Clear and single gene function: For the first time, Aspergillus0G048390, Aspergillus0G044330 and Aspergillus0G004350 were identified and verified as key functional genes in the dehydrogenation, re-dehydrogenation and thiolysis steps of fatty acid β-oxidation in Aspergillus nidulans. Individual overexpression can specifically enhance the efficiency of the corresponding catalytic steps and avoid metabolic flux disorder that may be caused by co-expression of multiple genes.

[0035] (2) Significant yield increase: All three single-gene engineered strains achieved a significant increase in the yield of echinocandin B. Specifically, the yield of OE-0G048390 in shake-flask fermentation was 2765.55±34.65 mg / L, which was 33.55% higher than that of the original strain (2070.75±40.05 mg / L); the yield of OE-0G044330 was 2591.25±25.95 mg / L, which was 25.14% higher; and the yield of OE-0G04350 was 2601.35±71.05 mg / L, which was 25.62% higher. These results were significantly better than existing technologies.

[0036] (3) The strain is easy to construct and has strong genetic stability: the single gene overexpression vector is easy to construct and has high transformation efficiency. After five consecutive generations, the fermentation performance of the engineered strain is stable, which meets the requirements of large-scale application in industrial production.

[0037] (4) Flexible application and wide adaptability: The three single genes can be flexibly selected and applied according to the type of fatty acid substrate (different carbon chain length) in the fermentation system. They can also be used as basic elements for subsequent multi-gene combination modification, providing technical support for the stepwise optimization of ECB production strains. At the same time, they can be extended to the modification of other secondary metabolite synthesis strains that depend on acetyl-CoA. Attached Figure Description

[0038] Figure 1 Plasmid maps of the recombinant overexpression vectors pDht-sk-0G048390, pDht-sk-0G044330, and pDht-sk-0G004350.

[0039] Figure 2 The results of PCR identification of recombinant plasmids pDht-sk-0G048390, pDht-sk-0G044330, and pDht-sk-0G004350 are shown.

[0040] Figure 3 The results show the increased fermentation yield of the strain. Detailed Implementation

[0041] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0042] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0043] Furthermore, it should be understood that the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, does not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, unless otherwise stated. It should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in this invention does not preclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned devices / apparatus, unless otherwise stated. Moreover, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or limiting the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0044] Culture medium and main reagents:

[0045] The Aspergillus nidulans ZJB16068 involved in the embodiments of this application was screened according to the screening method described in the literature "Eating the mechanisms of echinocandin B biosynthesis under fatty acid feeding in Aspergillus nidulans based on genome and transcriptome sequencing, 3 Biotech, 2025".

[0046] The components of LB liquid medium are as follows: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L NaCl.

[0047] The components of LB solid medium are as follows: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, and 10 g / L agar.

[0048] The components of YPD liquid culture medium are as follows: yeast extract 10 g / L, tryptone 20 g / L, glucose 20 g / L.

[0049] PDA medium: 200 g / L peeled potato, 20 g / L glucose, 20 g / L agar.

[0050] The components of CD medium are as follows: NaNO3 3 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, KCl 0.5 g / L, FeSO4•7H2O 0.01 g / L, glucose 30 g / L, and agar 15 g / L.

[0051] The components of the IM liquid culture medium are as follows: NaCl 0.3 g / L, NH4Cl 0.34 g / L, NaNO3 0.47 g / L, KH2PO4 0.136 g / L, MgSO4·7H2O 0.6 g / L, CaCl2·2H2O 0.01 g / L, and trace element supplement solution 1 mL. After sterilization, add 1 mL of nutrient supplement solution, 1 mL of 40 mM MES buffer, and 100 μL of 200 μM acetylsylgenone solution to the laminar flow hood.

[0052] The components of the IM solid culture medium are as follows: NaCl 0.3 g / L, NH4Cl 0.34 g / L, NaNO3 0.47 g / L, KH2PO4 0.136 g / L, MgSO4·7H2O 0.6 g / L, CaCl2·2H2O 0.01 g / L, trace element supplement 1 mL, and agar 20 g / L. After sterilization, 1 mL of nutrient supplement, 1 mL of 40 mM MES buffer, and 100 μL of 200 μM acetylsylgenone solution are added to the laminar flow hood.

[0053] The components of the trace element supplement solution are as follows: ZnSO4·7H2O 0.1 g / L, CuSO4·H2O 0.1 g / L, H3BO4 0.1 g / L, Na2MOO4·7H2O 0.1 g / L, with water as the solvent and natural pH.

[0054] The nutritional supplement solution consists of the following components: 200 g / L glucose solution, 500 g / L glycerin, water as solvent, and natural pH.

[0055] 40 mM MES buffer: Weigh 0.1562 g MES and dissolve it in 20 mL of water. Filter and sterilize in a clean bench.

[0056] 200μM acetylsuccinone (AS) solution: Weigh 0.786 g AS and dissolve it in 20 mL DMSO. Filter and sterilize in a clean bench.

[0057] 50 mg / mL kanamycin resistance solution: Weigh 0.5 g of kanamycin and dissolve it in 10 mL of water. Filter and sterilize in a clean bench.

[0058] 20 mg / mL rifampicin resistance solution: Weigh 0.5 g of rifampicin and dissolve it in 25 mL of DMSO. Filter and sterilize in a clean bench.

[0059] 200 mM cefotaxime sodium resistance solution: Weigh 1.91 g of cefotaxime and dissolve it in 20 mL of water. Filter and sterilize in an ultra-clean workbench.

[0060] The cell lysis buffer consisted of the following components: Na2EDTA·2H2O 23.263 g / L, Tris 7.575 g / L, SDS 30 g / L, pH 8.0.

[0061] The seed culture medium consists of the following components: glucose 10 g / L, glycerol 10 g / L, soybean meal 25 g / L, and natural pH.

[0062] The fermentation medium consisted of the following components: 70% methyl oleate 90 g / L, Tween 80 10.98 g / L, sucrose 9.44 g / L, peanut oil 20 g / L, glycerol 10 g / L, soybean meal 40 g / L, sucrose 9.44 g / L, tryptone 8.6 g / L, threonine 3.1 g / L, ornithine hydrochloride 6.1 g / L, K₂HPO₄ 6.1 g / L, MgSO₄·7H₂O 0.5 g / L, MnSO₄·H₂O 0.2 g / L, FeSO₄·7H₂O 0.05 g / L, CaCl₂ 0.3 g / L, CuSO₄·5H₂O 0.6 g / L, pH natural.

[0063] Example 1: Construction of recombinant overexpression vectors pDht-sk-0G048390, pDht-sk-0G044330, and pDht-sk-0G004350

[0064] (1) Genomic DNA extraction: Aspergillus nidulans ZJB16068 was inoculated into PDA medium and cultured at 25℃ for 7 days. Genomic DNA was extracted using a genomic DNA extraction kit (Solepro, D2300-100T). DNA integrity was verified by 1% agarose gel electrophoresis. The A260 / A280 ratio was 1.8-2.0 as determined by Nanodrop, indicating that the purity was qualified.

[0065] (2) Obtaining the Aspergillus0G048390, Aspergillus0G044330, and Aspergillus0G004350 genes: Using the genome of Aspergillus nidulans ZJB1606 as a template, the Aspergillus0G048390 gene fragment was obtained by PCR amplification using primers 48390-F and 48390-R, the Aspergillus0G044330 gene fragment was obtained by PCR amplification using primers 44330-F and 44330-R, and the Aspergillus0G004350 gene fragment was obtained by PCR amplification using primers 04350-F and 04350-R. The PCR amplification conditions were as follows: 98℃, 5 min; 98℃, 30 s; 58℃, 30 s; 72℃, 1 min 15 s, 32 cycles; 72℃, 5 min, 50 μL system.

[0066] (3) Obtaining the pDht-sk vector backbone: The plasmid pDht-sk was digested with restriction endonuclease XbaⅠ (both purchased from New England Biolabs (Beijing) LTD.), and the target band was recovered by electrophoresis to obtain the linearized plasmid backbone fragment. The PCR product was detected by 1.0% agarose gel electrophoresis and the fragment was excised and purified.

[0067] (4) Obtain the recombinant vectors pDht-sk-0G048390, pDht-sk-0G044330, and pDht-sk-0G004350: such as Figure 1 As shown, the Aspergillus0G048390, Aspergillus0G044330, and Aspergillus0G004350 genes were ligated with single-enzyme-digested linearized plasmid backbone fragments using a ClonExpress II One Step Cloning Kit (Vazyme) to construct overexpression vectors. The ligation products were transformed into E. coli DH5α and plated on LB agar plates containing 100 mg / L kanamycin resistance and incubated at 37°C for 12 h. Single colonies were randomly selected for PCR verification. Using gpdA-Ce-F1 and TtrpC-SC-R as upstream and downstream primers, the ligation nodes in the three recombinant plasmids were identified. Figure 2The primers used for PCR identification amplified gene fragments of 3364 bp, 2173 bp, and 2782 bp, respectively (the labeled lanes indicate the size of the gene fragments). Plasmids were extracted from correctly identified transformants and sequenced to obtain recombinant plasmids. Single colonies were picked and inoculated into LB broth (containing kanamycin), cultured at 37℃ and 220 rpm for 8 h, and the plasmids were extracted for sequencing verification, yielding the correct recombinant overexpression vectors pDht-sk-0G048390, pDht-sk-0G044330, and pDht-sk-0G004350. The PCR amplification conditions were as follows: 98℃ for 5 min; 98℃ for 30 s; 58℃ for 30 s; 72℃ for 1 min, 32 cycles; 72℃ for 5 min, 25 μL system.

[0068] Table 1: Primer sequences Primer sequence(5’→3’) gpdA-Ce-F1 accggtgactctttctggca TtrpC-SC-R ctcgtaggtctcttgacgac 48390-F cccgcttgagcagacatcacaATGCCAAATCCACCTCCCGC 48390-R CGGTCGGCATCTACTtctagaTCACAGCTTGCTTTTAATCTC 44330-F cccgcttgagcagacatcacaATGTTTGCCCGTCAATCCGC 44330-R CCGGTCGGCATCTACTtctagaTTACTCGTGCGACCACTGAG 04350-F cccgcttgagcagacatcacaATGGCATCAGATTCGACAGC 04350-R CGATCCGGTCGGCATCTACTtctagaTTACTCCCTAACCCAGACAG .

[0069] Example 2: Construction and Validation of Engineered Bacteria with Single Gene Overexpression

[0070] (1) Agrobacterium transformation: Take each recombinant plasmid (1 μg) and add it to Agrobacterium AGL1 competent cells. Incubate on ice for 5 min, freeze in liquid nitrogen for 5 min, incubate in water at 28℃ for 5 min, incubate on ice again for 5 min, add 700 μL LB liquid medium, and incubate at 28℃ and 200 rpm for 2-3 h. Spread on LB plates containing rifampicin (25 μg / mL) and kanamycin (50 μg / mL), and incubate at 28℃ for 48 h. Pick single colonies and use gpdA-Ce-F1 and TtrpC-SC-R as primers for PCR verification. Confirm that the recombinant Agrobacterium was successfully constructed and named: AGL-0G048390, AGL-0G044330, and AGL-0G004350. The PCR amplification conditions were as follows: 98℃ for 10 min; 98℃ for 30 s; 58℃ for 30 s; 72℃ for 1 min, 32 cycles; 72℃ for 5 min, 25 μL system.

[0071] (2) Agrobacterium induction culture: Recombinant Agrobacterium AGL-0G048390, AGL-0G044330, and AGL-0G004350 were inoculated into LB liquid medium (containing a final concentration of 50 μg / mL kanamycin and 25 μg / mL rifampin), and cultured at 200 rpm for 2 days in a shaker at 28°C. 1.5 mL of the cultured Agrobacterium suspension was centrifuged at 4000 rpm for 10 min, and the bacterial pellet was resuspended in 1 mL of IM liquid medium (containing a final concentration of 200 μg / mL AS), centrifuged at 4000 rpm for 10 min, and the bacterial pellet was retained. The bacterial pellet was then resuspended again in 4 mL of IM liquid medium (containing a final concentration of 200 μg / mL AS), and cultured at 200 rpm in a shaker at 28°C for 6-8 h.

[0072] (3) Spore suspension preparation and germination: Aspergillus nidulans ZJB16068 bacterial suspension was inoculated into PDA solid medium and cultured in a 25℃ incubator in the dark for about 7 days until green spores appeared. Then, the spores were scraped off with 0.02% Tween 80 solution and diluted to a concentration of 10. 7 Approximately / mL. Resuspend the spores in 2mL of YPD liquid medium and incubate at 28℃ with shaking at 200 rpm for 6-8 h to obtain highly active germinating spores. At this stage, the germinating spores are more conducive to Agrobacterium infection, thus improving transformation efficiency. Co-culture transformation: Use a suspension of Aspergillus nidulans ZJB16068 spores (10... 7 Mix the bacterial culture (number / mL) with the recombinant Agrobacterium tumefaciens in a 1:1 volume ratio, spread evenly on 1M solid medium containing 200μM acetylsyleugenol, and co-culture at 25℃ for 2-3 days until white mycelia appear on the plate.

[0073] (4) Verification of engineered strains: Scrape the hyphae and spores after co-culture and spread them on PDA plates containing hygromycin (50 μg / mL) and cefotaxime sodium (200 μg / mL). Incubate at 25℃ for 5-7 days. Pick the Aspergillus nidulans transformants to extract genomic DNA. Use gpdA-Ce-F1 and TtrpC-SC-R as upstream and downstream primers for PCR identification. The expected length fragments (3364bp, 2173bp, 2782bp) were amplified, proving that the target gene has been integrated into the Aspergillus nidulans genome. The correct engineered strains OE-0G048390, OE-0G044330, and OE-0G004350 were obtained.

[0074] Example 3: Shake-flask fermentation of OE-0G048390, OE-0G044330, and OE-0G004350 overexpression engineered strains

[0075] (1) Plate culture of engineered strains: Spores of the three engineered strains obtained in Example 2 were obtained by streak plating. The spore suspension was serially diluted 100 times, and 10 µL of the diluted spore suspension was streaked on a PDA plate to obtain single colonies. The colonies were cultured in a 25℃ incubator in the dark for 7-10 days for seed culture.

[0076] (2) Seed culture: After 7-10 days of PDA plate culture, use a sterile inoculation spatula to take a 1 cm² single colony agar block of light green color and inoculate it into a 250 mL Erlenmeyer flask with 20% liquid volume. Incubate at 25℃ and 220 rpm for 3 days until the culture medium turns into an orange-red slightly thick state.

[0077] (3) Fermentation culture: Take 5 mL of seed liquid and inoculate it into a 250 mL Erlenmeyer flask with a 20% liquid volume. Incubate at 25℃ and 220 rpm for 10 days. The seed liquid inoculation volume is 10% (v / v). The 10 days is the whole fermentation cycle. During the period, the color of the fermentation broth changes from white to orange-yellow to orange-red to reddish-brown / dark brown.

[0078] Example 4: HPLC detection of echinocandin B:

[0079] (1) Sample preparation of fermentation broth: After shaking the fermentation broth, take 2 mL of fermentation broth into a 10 mL EP tube, add methanol, dilute 5 times, mix well, and let stand for extraction for 12-24 h (mix 2-3 times during the period). Take the supernatant, filter it through a 0.22 μm organic membrane, and then perform liquid chromatography analysis.

[0080] (2) Preparation of echinocandin B standard solution: Echinocandin B standard (GC>99%) was dissolved in methanol to prepare standard solutions of different concentrations (0.1 g / L, 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1.0 g / L). 1 mL of each solution was placed in a liquid chromatography bottle for liquid chromatography detection.

[0081] (3) Liquid chromatography detection method: The detection instrument was an Agilent 1260 high performance liquid chromatograph; the chromatographic column was a C18 column (Dalian Elite 4.6 mm × 250 mm × 5 μm); the mobile phase was methanol: acetonitrile: water = 7:1:2; the flow rate was 1 mL / min; the ultraviolet detection wavelength was 222 nm; the injection volume was 20 μL; and the column temperature was 40℃.

[0082] (4) The retention time of echinocandin B is approximately 18 min. A standard curve was plotted with peak area Y as the ordinate and concentration X (mg / L) as the abscissa. The regression equation was: Y = 11.92371X - 273.80881, R² = 0.99983. This indicates that within the concentration range of 100-1000 mg / L, there is a good linear correlation between the peak area and concentration of echinocandin B.

[0083] (5) Fermentation results are as follows Figure 3 As shown, the yield of strain OE-0G048390 during shake-flask fermentation was 2765.55±34.65 mg / L, an increase of 33.55% compared to the original strain (2070.75±40.05 mg / L); the yield of OE-0G044330 was 2591.25±25.95 mg / L, an increase of 25.14%; and the yield of OE-0G04350 was 2601.35±71.05 mg / L, an increase of 25.62%.

[0084] Example 5: Genetic stability analysis of OE-0G048390, OE-0G044330, and OE-0G004350 overexpression engineered strains

[0085] The correct transformants OE-0G048390, OE-0G044330, and OE-0G004350 from Example 3 were selected and passaged five times. They were then cultured on the same PDA solid medium as in Example 3. After the same culture method and HPLC analysis as in Examples 3 and 4, the dissolved oxygen increased during the fermentation process, and the yield of echinocandin B was stable, indicating that the echinocandin B-producing engineered strain constructed in this invention has passage stability.

[0086] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in methods and compositions, will be apparent to those skilled in the art without departing from the scope and spirit of this invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this invention, such as codon optimization of Aspergillus0G048390, Aspergillus0G044330, and Aspergillus0G004350, or homologous substitutions of homologous proteins (with similarity greater than 80%) of the Aspergillus0G048390, Aspergillus0G044330, and Aspergillus0G004350 genes, should be included within the scope of protection of this invention. Although this invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that this invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

[0087] SEQ ID NO:1:

[0088] SEQ ID NO:2: MPNPPPAWVQALKPASPQGTELLTQERAQSNIDVDTLGNLLHTKEALKKQDEILSVLKSEKVFDKSRNHVLGRTEKIQLALARGKRLQQLKKVHNWSDEDVHVANELVAEPTPYGLHASMFLVTLREQGTPEQHKLFYERARNYEIIGCYAQTELGHGSNVRGLETTATWDPSDQTFIIHSPSLTASKWWIGSLGRTANHAVVMAQLYIGGKNYGPHPFVVQIRDMETHQPLENVYVGDIGPKFGYNTMDNGFLLFNKLKIPHVNMLARFAQVDKATNKYLRPASPSLMYGTMTWVRSNIVLQAGGVLARGVTIAVRYCAVRRQFQDRDAKANAEENQVLNYKMVQIRLLPLLAAMYALHFTGRGMMRLYEENQERMKAAAQADQEKRGAGPEQLRAGSDLLADLHATSCGLKALASTTAGEGLEVCRRACGGHGYSNYSGIGPWYSDYLPTLTWEGDNYMLTQQVARYLLKSARAVLAGKGTANDTSRILQAYLARRDKGASFDILGDDADIVAAFAWRTAHLTFETLKYRDVEKRSWNSLLINFWRLSTALSQYLVVKNFYEAVNSPEIRSSLDKDTASTLRSLFRLYALHTLEREASEFFSSAAVTVRQIGLTQTSEVPKLLDEIRPHAVRLVDSWKIPDWQLDSALGRSDGDVYPDLFKRASMQNPVNDLVFDPYPWNENVLKNGGEIKSKL。

[0089] SEQ ID NO:3:

[0090] SEQ ID NO:4: MFARQSARFLFPRTTTVIARVRLYSSASPSYEHILTSTPKPGVGLITLNRPKALNALSSPLFKELNDALSKYDESKDIGAIIITGSEKAFAAGADIKEMAPLTFASAYSNNFIAPWSHLANSIRKPVIAAVSGYALGGGCELALMCDIIYCTASATFGQPEIKLGVIPGAGGSQRLTAAVGKSKAMELILTGKNFSGKEAGEWGVAAKVVDGGKEELLEEAVKTAETIAGYSRVATVAAKEVVNKSQDLGVREGVEYERRLFHGLFGSQDQKIGMTAFAEKKKPQWSHE.

[0091] SEQ ID NO:5:

[0092] SEQ ID NO:6: MTVINEEAISSPILQEINNIKNQFKVDTASLKHITDHFVCQLEDGLARYDGEIPMNVTWVPSFPTGYETGRYLAIDMGGTNLRICDVTLTEEKGAYTIEQDKYRLPIHLRKGKGAELWEFIAAKLEDFLVKHKLAREDGEKLPLAFTFSYPVTQDHIRHGVLQRWTKGFDISGVEGEDVVAHLEEVFEKRNVPVRLVALVNDTVGTLIASAYKNPAIKIGSIFATGCNAAYMEKVSRIPKITDHGSEFESDALVSINCEYGAFDNGHKVLPMTRFDEEIDQTSARPGQQAYEKMVAGMYMGELLRLLLLHLHESSGFFTDAEIDRLRGYGTMDSASLSRMEAGGSEAERMADTKCILKELYGIEATDEEARACCLLGEIVCTRAARLYACGIAALCRKQGIDECAVGVDGSTFEKYSQFRERAADALGEILDWPERQQLVKLVTAEDGSGVGSALIAAITLNQ。

Claims

1. A gene encoding the fatty acid β-oxidation pathway derived from Aspergillus nidulans, characterized in that, The encoding gene is selected from the group consisting of: (a) the Aspergillus0G048390 gene with the nucleotide sequence as shown in SEQ ID NO:1; (b) the Aspergillus0G044330 gene with the nucleotide sequence as shown in SEQ ID NO:3; and / or (c) the Aspergillus0G004350 gene with the nucleotide sequence as shown in SEQ ID NO:

5.

2. The protein encoded by the fatty acid β-oxidation pathway encoding gene derived from Aspergillus nidulans as described in claim 1, characterized in that, The amino acid sequences of the proteins are shown in SEQ ID NO:2, SEQ ID NO:4 and / or SEQ ID NO:6; wherein SEQ ID NO:2 corresponds to the protein encoded by the Aspergillus0G048390 gene with the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4 corresponds to the protein encoded by the Aspergillus0G044330 gene with the nucleotide sequence shown in SEQ ID NO:3, and SEQ ID NO:6 corresponds to the protein encoded by the Aspergillus0G004350 gene with the nucleotide sequence shown in SEQ ID NO:

5.

3. A recombinant overexpression vector, characterized in that, It includes the gene encoding the fatty acid β-oxidation pathway derived from Aspergillus nidulans as described in claim 1, as well as an operable promoter and terminator.

4. The recombinant overexpression vector according to claim 3, characterized in that, The recombinant overexpression vector was obtained by inserting a gene encoding the fatty acid β-oxidation pathway from Aspergillus nidulans as the base vector. The gene is driven by the constitutive promoter gpdA and terminated by the terminator TtrpC.

5. A genetically engineered bacterium containing the coding gene of claim 1 or the recombinant overexpression vector of claim 3.

6. The genetically engineered bacterium according to claim 5, characterized in that, The genetically engineered bacteria are Aspergillus nidulans, which produces echinocandin B, as the host bacteria. The recombinant overexpression vector is transformed into the host bacteria to obtain engineered strains that overexpress the Aspergillus0G048390 gene, the Aspergillus0G044330 gene, and / or the Aspergillus0G004350 gene.

7. The method for constructing genetically engineered bacteria according to claim 5, characterized in that, Includes the following steps: (1) The coding genes for the fatty acid β-oxidation pathway were screened from the genome of Aspergillus nidulans ZJB16068; the coding genes are Aspergillus0G048390, Aspergillus0G044330 and / or Aspergillus0G004350. (2) Using the Escherichia coli-Agrobacterium shuttle plasmid pDht-sk as the basic vector, recombinant overexpression vectors containing the Aspergillus0G048390 gene, the Aspergillus0G044330 gene, and the Aspergillus0G004350 gene were constructed respectively. (3) The constructed recombinant overexpression vector was transformed into Aspergillus nidulans ZJB16068 using Agrobacterium AGL1-mediated transformation method to obtain engineered strains that overexpress the Aspergillus0G048390 gene, the Aspergillus0G044330 gene, or the Aspergillus0G004350 gene.

8. The application of the encoding gene of claim 1, the protein of claim 2, the recombinant overexpression vector of claim 3 or 4, or the genetically engineered bacteria of claim 5 or 6 in the fermentation production of echinocandin B.

9. A method for producing echinocandin B, characterized in that, The genetically engineered bacteria described in claim 5 or 6 are inoculated into a fermentation medium for fermentation culture to obtain echinocandin B.

10. The production method according to claim 8, characterized in that, The fermentation conditions were as follows: temperature 25℃, rotation speed 200 r / min, fermentation cycle 10 h; fermentation medium consisted of 70% methyl oleate 90 g, Tween 80 10.9 g, sucrose 9.4 g, peanut oil 20 g, glycerol 10 g, soybean meal 40 g, sucrose 9.4 g, tryptone 8.6 g, threonine 3.1 g, ornithine hydrochloride 6.1 g, K2HPO4 6.1 g, MgSO4·7H2O 0.5 g, MnSO4·H2O 0.2 g, FeSO4·7H2O 0.05 g, CaCl2 0.3 g, CuSO4·5H2O 0.6 g, and natural pH.

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