Establishing an Efficient Genetic Manipulation System for Sulfated Echinocandin Producing Fungus Coleophoma empetri

Men, Ping; Wang, Min; Li, Jinda; Geng, Ce; Huang, Xiaowan; Lü, Xuefeng

doi:10.3389/fmicb.2021.734780

Cited by 10 publications

(12 citation statements)

References 31 publications

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“…However, in their research, the efficiency of gene editing was not studied. Recently, Men et al reported protoplast-mediated transformation, and this method was used to achieve the disruption of the melanin gene . In their study, the gene disruption efficiency was only 4%, which demonstrated a low efficiency of the traditional gene editing method in C.…”

Section: Introductionmentioning

confidence: 99%

Analysis of FR901379 Biosynthetic Genes in Coleophoma empetri by Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Based Genomic Manipulation

et al. 2022

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The compound FR901379, a sulfated echinocandin produced by the filamentous fungus Coleophoma empetri F-11899, is an important intermediate for the synthesis of the antifungal drug micafungin. In this study, we established an efficient clustered regularly interspaced short palindromic repeats/Cas9-based gene editing tool for the industrial production strain C. empetri SIPI1284. With this method, the efficiency of gene mutagenesis in the target locus is up to 84%, which enables the rapid gene disruption for the analysis of FR901379 biosynthetic genes. Next, we verified the putative functional genes of the FR901379 biosynthetic gene cluster via gene disruption and gene complementation in vivo. These core functional genes included the nonribosomal peptide synthetase gene (CEnrps), the fatty-acyl-AMP ligase gene (CEligase) responsible for the formation of the activated form of palmitic acid and its transfer to CEnrps, four nonheme mononuclear iron oxygenase genes (CEoxy1, CEoxy2, CEoxy3, and CEoxy4) responsible for the synthesis of nonproteinogenic amino acids, l-homotyrosine biosynthesis genes (CEhtyA-D), two cytochrome P450 enzyme genes (CEp450-1 and CEp450-2), and a transcription regulator gene (CEhyp). In addition, by screening the whole genome, we identified two unknown genes (CEp450-3 and CEsul) responsible for the sulfonyloxy group of FR901379, which were separated from the core FR901379 biosynthetic cluster. Furthermore, during gene disruptions in the research, we obtained a series of FR901379 analogues and elucidated the relationship between the groups and antifungal activities.

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Section: Introductionmentioning

confidence: 99%

Analysis of FR901379 Biosynthetic Genes in Coleophoma empetri by Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Based Genomic Manipulation

et al. 2022

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“…All of the expression cassettes were individually introduced into C. empetri MEFC09 through (PEG)-CaCl 2 -mediated protoplast transformation [ 34 ]. Transformants were selected on PDAS plates amended with 100 mg/L hygromycin B and verified by genomic PCR.…”

Section: Methodsmentioning

confidence: 99%

“…The putative function of the predicted enzymes was confirmed with the online NCBI BLASTP programmer ( http://blast.ncbi.nlm.nih.gov ). Gene disruption was carried out via homologous recombination as described previously [ 34 ]. To knock out the CEfks1 gene, approximately 1.2 kb of 5' and 3' DNA of the CEfks1 gene were amplified by PCR from the genome of C. empetri MEFC09 using the primer pairs UCEfks1-F/UCEfks1-R and DCEfks1-F/DCEfks1-R and were fused with the marker hph by fusion PCR.…”

Section: Methodsmentioning

confidence: 99%

“…For the fermentation of all C. empetri strains, the fresh mycelia were crushed and inoculated in 50 mL of seed medium MKS (soluble starch 15 g/L, sucrose 10 g/L, cottonseed meal 5 g/L, peptone 10 g/L, KH 2 PO 4 1 g/L, and CaCO 3 2 g/L; pH 6.5) in 250 mL shake flasks for 2 days at 25 °C and 220 rpm [ 34 ]. Then, 5 mL of seed culture was inoculated into 50 mL of fermentation medium MKF (glucose 10 g/L, corn starch 30 g/L, peptone 10 g/L, D -sorbitol 160 g/L, (NH 4 ) 2 SO 4 6 g/L, KH 2 PO 4 1 g/L, FeSO 4 ·7H 2 O 0.3 g/L, ZnSO 4 ·7H 2 O 0.01 g/L, and CaCO 3 2 g/L; pH 6.5) and cultivated at 25 °C and 220 rpm for 8 days.…”

Section: Methodsmentioning

confidence: 99%

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Improving the production of the micafungin precursor FR901379 in an industrial production strain

et al. 2023

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Background Micafungin is an echinocandin-type antifungal agent used for the clinical treatment of invasive fungal infections. It is semisynthesized from the sulfonated lipohexapeptide FR901379, a nonribosomal peptide produced by the filamentous fungus Coleophoma empetri. However, the low fermentation efficiency of FR901379 increases the cost of micafungin production and hinders its widespread clinical application. Results Here, a highly efficient FR901379-producing strain was constructed via systems metabolic engineering in C. empetri MEFC09. First, the biosynthesis pathway of FR901379 was optimized by overexpressing the rate-limiting enzymes cytochrome P450 McfF and McfH, which successfully eliminated the accumulation of unwanted byproducts and increased the production of FR901379. Then, the functions of putative self-resistance genes encoding β-1,3-glucan synthase were evaluated in vivo. The deletion of CEfks1 affected growth and resulted in more spherical cells. Additionally, the transcriptional activator McfJ for the regulation of FR901379 biosynthesis was identified and applied in metabolic engineering. Overexpressing mcfJ markedly increased the production of FR901379 from 0.3 g/L to 1.3 g/L. Finally, the engineered strain coexpressing mcfJ, mcfF, and mcfH was constructed for additive effects, and the FR901379 titer reached 4.0 g/L under fed-batch conditions in a 5 L bioreactor. Conclusions This study represents a significant improvement for the production of FR901379 and provides guidance for the establishment of efficient fungal cell factories for other echinocandins.

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“…The fungi Coleophoma cylindrospora or Coleophoma empetri F-11899 produces the NRP acylated to fatty acid, coded as FR901379 (16) . This sulfated echinocandin is a precursor of the partial chemical synthesis of the echinocandin, micafungin, commercially sold as Mycamine and Fungiguard [ 103 ]. Recently, the BGCs involved in the biosynthesis of FR901379 (16) were analyzed and an efficient clustered regularly interspaced short palindromic repeats/Cas9-based gene editing tool for the industrial production strain C. empetri SIPI1284 was established [ 104 ].…”

Section: Bioactive Fungal Secondary Metabolites and Derivatives Succe...mentioning

confidence: 99%

Overview of Bioactive Fungal Secondary Metabolites: Cytotoxic and Antimicrobial Compounds

et al. 2022

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Microorganisms are known as important sources of natural compounds that have been studied and applied for different purposes in distinct areas. Specifically, in the pharmaceutical area, fungi have been explored mainly as sources of antibiotics, antiviral, anti-inflammatory, enzyme inhibitors, hypercholesteremic, antineoplastic/antitumor, immunomodulators, and immunosuppressants agents. However, historically, the high demand for new antimicrobial and antitumor agents has not been sufficiently attended by the drug discovery process, highlighting the relevance of intensifying studies to reach sustainable employment of the huge world biodiversity, including the microorganisms. Therefore, this review describes the main approaches and tools applied in the search for bioactive secondary metabolites, as well as presents several examples of compounds produced by different fungi species with proven pharmacological effects and additional examples of fungal cytotoxic and antimicrobial molecules. The review does not cover all fungal secondary metabolites already described; however, it presents some reports that can be useful at any phase of the drug discovery process, mainly for pharmaceutical applications.

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Establishing an Efficient Genetic Manipulation System for Sulfated Echinocandin Producing Fungus Coleophoma empetri

Cited by 10 publications

References 31 publications

Analysis of FR901379 Biosynthetic Genes in Coleophoma empetri by Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Based Genomic Manipulation

Analysis of FR901379 Biosynthetic Genes in Coleophoma empetri by Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Based Genomic Manipulation

Improving the production of the micafungin precursor FR901379 in an industrial production strain

Overview of Bioactive Fungal Secondary Metabolites: Cytotoxic and Antimicrobial Compounds

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