Inffumafungin analogue Asepticfungin with antifungal activity as well as preparation engineering bacteria and application thereof

Abstract

The invention discloses an inffumafungin analogue AsepticFungin with antifungal activity as well as preparation engineering bacteria and application of the inffumafungin analogue AsepticFungin. A series of AsepticFungin triterpenoid glycoside products with novel structures are obtained through fermentation and separation by virtue of a microbial heterologous expression platform, and AsepticFungin compounds with antifungal activity are screened out; the obtained compound has specificity in antifungal activity and can specifically inhibit growth of candida glabrata, the activity of AseptuFunginB is better, 90% of candida glabrata can be killed under the dosage of 1 microgram / L, and the compound can be used as a candidate drug for later antifungal activity.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an infungin analogue, asepticufungin, having antifungal activity, and an engineered bacteria for its preparation and application. Background Art

[0002] Fungi are heterotrophic eukaryotic organisms with cell wall structures that are widely present in nature. Fungal infections can be divided into two categories based on the site of invasion of human tissues and organs: superficial fungal infections and deep fungal infections (invasive fungal infections). In recent years, the incidence of invasive fungal infections (IFIs) has been increasing year by year due to the increasing number of people with weakened immune systems (such as the elderly, AIDS patients, and cancer patients undergoing radiotherapy and chemotherapy), the increasing number of people requiring immunosuppressants (such as organ transplant recipients), and the overuse of broad-spectrum antibiotics and glucocorticoids.

[0003] Due to the high morbidity and mortality rates of invasive fungal infections in clinical practice, as well as the emergence and increase in drug-resistant fungal strains and highly virulent mutant fungal strains, the treatment of invasive fungal infections is relatively difficult. The limited range of antifungal drugs available in clinical practice is a major bottleneck in the treatment of invasive fungal infections. Currently, commonly used drugs for the treatment and prevention of invasive fungal infections can be divided into four major categories: polyenes, pyrimidines, echinocandins, and azoles. These antifungal drugs all have certain limitations, mainly manifested in limited clinical efficacy, narrow antifungal spectrum, poor pharmacokinetic characteristics, significant side effects, and drug-drug interactions. Therefore, the search for novel antifungal targets and related drugs is imminent.

[0004] Ibrexafungerp (erefungin) was approved by the FDA in 2021 for the clinical treatment of invasive vulvar candidiasis and is the first new antifungal drug approved in nearly two decades. Ibrexafungerp destroys the formation of fungal cell walls by nonspecific competitive inhibition of β-1,3-glucan synthase (GS), which is a fungus-specific target. Compared with the above four types of antifungal drugs, Ibrexafungerp has (1) broad-spectrum antibacterial activity, with antibacterial activity against Aspergillus and Candida; (2) antibacterial activity against multiple drug-resistant strains; (3) high bioavailability, can be taken orally or injected; (4) safe and well-tolerated, with a long half-life and high tissue penetration. Currently, Ibrexafungerp is mainly obtained by chemical semi-synthesis of the natural product Enfumafungin, but the original production strain synthesizes Enfumafungin with low yield and difficult fermentation separation, which limits the development of Ibrexafungerp. In 2023, Hu Dan's research group reported that fuscoatroside, an infumafungin analogue, also possesses antifungal activity. Therefore, identifying more infumafungin analogues is an effective prerequisite and important guarantee for the development of new antifungal drugs. Summary of the Invention

[0005] In view of this, one of the objects of the present invention is to provide an engineered bacterium for preparing an asepticufungin compound; a second object of the present invention is to provide an antifungal active compound asepticufungin synthesized by the engineered bacterium; and a third object of the present invention is to provide the use of the antifungal active compound asepticufungin in the preparation of antifungal drugs.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] 1. An engineered bacterium for preparing an asepticufungin compound, the engineered bacterium being constructed by using Aspergillus nidulans as a starting strain and recombinantly expressing the Asp1 gene, Asp2 gene, Asp3 gene, and Asp4 gene from Aspergillus ellipticus CBS 707.79, or recombinantly expressing the Asp1 gene, Asp2 gene, Asp3 gene, Asp4 gene, and Asp5 gene from Aspergillus ellipticus CBS 707.79; the nucleotide sequence of the Asp1 gene is as set forth in SEQ ID NO. 15, the nucleotide sequence of the Asp2 gene is as set forth in SEQ ID NO. 16, the nucleotide sequence of the Asp3 gene is as set forth in SEQ ID NO. 17, the nucleotide sequence of the Asp4 gene is as set forth in SEQ ID NO. 18, and the nucleotide sequence of the Asp5 gene is as set forth in SEQ ID NO. 19.

[0008] 2. An antifungal active compound synthesized by the engineered bacteria, named asepticufungin, characterized in that the general formula of the compound is shown in Formula I below:

[0009]

[0010] In some embodiments of the present invention, the compound is as follows:

[0011]

[0012] 3. Use of the antifungal active compound Asepticufungin in the preparation of antifungal drugs.

[0013] In some embodiments of the present invention, the fungus is Candida glabrata.

[0014] The beneficial effects of the present invention are as follows: the present invention obtains a series of novel structural asepticufungin triterpene glycoside products through fermentation and separation via a microbial heterologous expression platform, and screens out asepticufungin compounds with antifungal activity; the antifungal activity of the obtained compounds is specific and can specifically inhibit the growth of Candida glabrata, among which Asepticufungin B has the better activity and can kill 90% of Candida glabrata at a dose of 1 μg / L, and can be used as a candidate drug for later antifungal development. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0016] Figure 1This is the PIM001 plasmid map.

[0017] Figure 2 This is the PIM002 plasmid map.

[0018] Figure 3 This is the PIM003 plasmid map.

[0019] Figure 4 This is the structure of Asepticufungin A.

[0020] Figure 5 This is the structure of Asepticufungin B. DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0022] Example 1. Preparation of Compound Asepticufungin A

[0023] Plasmid construction: The genome of Aspergillus ellipticus CBS 707.79 was extracted and used as a template. Genes Asp1, Asp2, Asp3, and Asp4 (terpenoid cyclase-glycosyltransferase, cytochrome P450-2, acetyltransferase, cytochrome P450-4, SEQ ID NOs. 15-18) were amplified by PCR using primers pANR-Asp1-F / pANR-Asp1-R, pANP-Asp2-F / pANP-Asp2-R, pANP-Asp3-F / pANP-Asp3-R, and pANP-Asp4-F / pANP-Asp4-R as shown in Table 1. The gpdA promoter was obtained by PCR using the plasmid pANR as a template and primers gpdA-F / gpdA-R. The glaA promoter was obtained by PCR using primers glaA-F / glaA-R as a template. The plasmid vectors pANR and pANP were linearized by BamH I. The Asp1 fragment and the linearized pANR vector were co-transfected into the yeast BJ5464-NpgA to obtain the recombinant plasmid pANR-Asp1 (abbreviated as pIM001, Figure 1 ), the Asp2, Asp3, Asp4 fragments, promoters gpdA, glaA and the linearized vector of pANP were co-transformed into Saccharomyces cerevisiae BJ5464-NpgA to obtain the recombinant plasmid pANP-Asp234 (abbreviated as pIM002, Figure 2 ).

[0024] Table 1 Primer sequences

[0025]

[0026]

[0027] Preparation of Asepticufungin A: Genes Asp1, Asp2, Asp3, and Asp4 (terpenoid cyclase-glycosyltransferase, cytochrome P450-2, and acetyltransferase, cytochrome P450-4, SEQ ID NOs. 15-18) from Aspergillus ellipticus CBS 707.79 were heterologously expressed in Aspergillus nidulans. The heterologous expression strain was fermented in large quantities. After fermentation, the strain was extracted three times with acetone:ethyl acetate (1:3) and concentrated to dryness under reduced pressure. The first fraction was separated using medium-pressure preparative chromatography using a methanol-water solvent and an elution profile of 45% methanol for 20 minutes, 45%-100% methanol for 40 minutes, and 100% methanol for 20 minutes. Fractions containing asepticufungin A were combined. Preparative liquid chromatography (column model: YMC ODS-A 5μm 120A (10×250mm)) was then used for separation, with isocratic elution using 85% acetonitrile-water, to obtain 14 mg of pure asepticufungin A. Asepticufungin A was dissolved in deuterated pyridine and identified by nuclear magnetic resonance. The compound identities are shown in Table 2.

[0028] Asepticufungin A identification: white powder; HR TOFMS m / z 683.4536 [M+Na] in positive ion mode + ,C 38 H 60 The calculated value of O9Na is 683.4135, indicating that the molecular formula of the compound is C 38 H 60 O9, combined with the NMR data in Table 2, the structure of the compound was determined, as shown in Figure 4 shown.

[0029] Table 2 NMR data assignment of Asepticufungin A

[0030]

[0031]

[0032] (400MHz for 1 H NMR, 100 MHz for 13 C NMR)

[0033] Example 2. Preparation of Compound Asepticufungin B

[0034] Plasmid construction: The genome of Aspergillus ellipticus CBS 707.79 was extracted and used as a template. The Asp5 gene (cytochrome P450-5, SEQ ID NO. 19) was amplified by PCR using primers pANU-Asp5-F / pANU-Asp5-R. The plasmid vector pANU was linearized by Not I digestion. The Asp5 fragment and the linearized pANU vector were co-transfected into Saccharomyces cerevisiae BJ5464-NpgA to obtain the recombinant plasmid pANU-Asp5 (abbreviated as pIM003, Figure 3 ).

[0035] Preparation of Asepticufungin B: Genes Asp1, Asp2, Asp3, Asp4, and Asp5 (terpenoid cyclase-glycosyltransferase, cytochrome P450-2, acetyltransferase, cytochrome P450-4, and cytochrome P450-5, SEQ ID NOs. 15-19) from Aspergillus ellipticus CBS 707.79 were heterologously expressed in Aspergillus nidulans. The heterologous expression strain was fermented in large quantities. After fermentation, the product was extracted three times with acetone:ethyl acetate (1:3) and concentrated to dryness under reduced pressure. The product was initially separated using medium-pressure preparative chromatography using a methanol-water solvent and an elution profile of 40% methanol for 20 minutes, 40%-100% methanol for 40 minutes, and 100% methanol for 20 minutes. Fractions containing asepticufungin B were combined. Subsequently, separation was performed using a preparative liquid chromatography column (YMC ODS-A 5μm 120A (10×250mm)) and isocratic elution with 70% acetonitrile-water to obtain 7 mg of pure asepticufungin B. Asepticufungin B was dissolved in deuterated methanol and identified by nuclear magnetic resonance. The compound identities are shown in Table 3.

[0036] Asepticufungin B identification: white powder; HR TOFMS m / z 689.4429 [MH] in negative ion mode - ,C 38 H 58 O 11 The calculated value is 690.8710, indicating that the molecular formula of the compound is C 38 H 58 O 11 , combined with the NMR data in Table 3, the structure of the compound is determined as Figure 5 shown.

[0037] Table 3 NMR data assignment of Asepticufungin B

[0038]

[0039]

[0040] (400MHz for 1 H NMR, 100 MHz for 13 C NMR)

[0041] Example 3: Antifungal Effect of Asepticufungin Compounds

[0042] Compounds A and B in Examples 1 and 2 were subjected to antifungal experiments. The specific method is as follows: RPMI1640 medium was inoculated with seven species of Candida, namely Candida albicans, Candida auris, Candida tropicalis, Candida glabrata, Candida kruseii, Candida parapsilosis, and Cryptococcus neoformans, with an inoculum size of 5000 cells / plate. The cells were cultured at 37 degrees Celsius for 24 hours. The above-mentioned compounds A and B were prepared into 12 concentrations (μg / mL): 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0. The compounds were dissolved in DMSO and then treated with the compounds to observe the antifungal activity of Candida. The statistical results are shown in Table 3.

[0043] Table 3 Antifungal effect of Asepticufungin compounds

[0044]

[0045]

[0046] The results showed that both compound A and compound B had moderate antifungal effects, and both had stronger antifungal effects against Candida glabrata. Compound B was more effective, with an MIC of 1 μg / L against Candida glabrata.

[0047] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. An engineered bacterium for preparing an asepticufungin compound, characterized in that: The engineered bacteria is constructed by using Aspergillus nidulans as a starting strain and recombinantly expressing the Asp1 gene, Asp2 gene, Asp3 gene and Asp4 gene from Aspergillus ellipticus CBS 707.79, or recombinantly expressing the Asp1 gene, Asp2 gene, Asp3 gene, Asp4 gene and Asp5 gene from Aspergillus ellipticus CBS 707.79; the nucleotide sequence of the Asp1 gene is as described in SEQ ID NO.15, the nucleotide sequence of the Asp2 gene is as described in SEQ ID NO.16, the nucleotide sequence of the Asp3 gene is as described in SEQ ID NO.17, the nucleotide sequence of the Asp4 gene is as described in SEQ ID NO.18, and the nucleotide sequence of the Asp5 gene is as described in SEQ ID NO.

19.

2. An antifungal active compound synthesized by the engineered bacteria according to claim 1, named asepticufungin, characterized in that: The general formula of the compound is shown in Formula I below:

3. Use of the antifungal compound asepticufungin according to claim 2 in the preparation of antifungal drugs.

4. The use according to claim 3, characterized in that: The fungus is Candida glabrata.

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