Establishing an efficient gene-targeting system in an itaconic-acid producing Aspergillus terreus strain

Huang, Xiaowan; Chen, Mei; Li, Jianjun

doi:10.1007/s10529-016-2143-y

Cited by 18 publications

(15 citation statements)

References 27 publications

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“…( Ninomiya et al, 2004 ). However, homologous recombination (HR) efficiency in filamentous fungi is extremely low because the NHEJ pathway dominates the repair of DNA double-strand breaks ( Huang et al, 2016 ). Learning from previous research, we tried to improve the frequency of gene targeting by disrupting the NHEJ pathway.…”

Section: Resultsmentioning

confidence: 99%

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

Men

Wang

et al. 2021

Front. Microbiol.

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Micafungin is an important echinocandin antifungal agent for the treatment of invasive fungal infections. In industry, micafungin is derived from the natural product FR901379, which is a non-ribosomal cyclic hexapeptide produced by the filamentous fungus Coleophoma empetri. The difficulty of genetic manipulation in C. empetri restricts the clarification of FR901379 biosynthetic mechanism. In this work, we developed an efficient genetic manipulation system in the industrial FR901379-producing strain C. empetri MEFC009. Firstly, a convenient protoplast-mediated transformation (PMT) method was developed. Secondly, with this transformation method, the essential genetic elements were verified. Selectable markers hph, neo, and nat can be used for the transformation, and promotors Ppgk, PgpdA, and PgpdAt are functional in C. empetri MEFC009. Thirdly, the frequency of homologous recombination was improved from 4 to 100% by deleting the ku80 gene, resulting in an excellent chassis cell for gene-targeting. Additionally, the advantage of this genetic manipulation system was demonstrated in the identification of the polyketide synthase (PKS) responsible for the biosynthesis of dihydroxynapthalene (DHN)-melanin. This genetic manipulation system will be a useful platform for the research of FR901379 and further genome mining of secondary metabolites in C. empetri.

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Section: Resultsmentioning

confidence: 99%

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

Men

Wang

et al. 2021

Front. Microbiol.

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“…A. terreus HZ01 is an industrial lovastatin-producing strain that was provided by Hisun Pharm. The ku80 – / pyrG – double mutant HZ03, which is a chassis cell used for the following gene-targeting and marker rescue operations, was constructed as previously described . The cell growth and lovastatin productivity of HZ02 were assessed to evaluate the influences of the ku80 deletion.…”

Section: Methodsmentioning

confidence: 99%

“…For the next round of deletion of lovF , it was necessary to perform the marker rescue in advance. The selectable marker loxP-pyrG An -loxP of HZ-ΔlovF1 was excised using the modified Cre/ loxP recombination system as previously described, , resulting in the uracil auxotrophic A. terreus strain HZ-ΔlovF1-ΔpyrG An . The gene-targeting DNA elements of the remaining copies were constructed as lovF-KO1 using the corresponding primers listed in Table S1, and used for multiple rounds of lovF deletion.…”

Section: Methodsmentioning

confidence: 99%

“…The regulator genes laeA and lovE were amplified from the cDNA of A. nidulans and A. terreus HZ01, respectively. The DNA fragments Uku80-pyrGAn and TtrpC-Dku80 were amplified from the plasmid pXH106 using the primer pairs U-ku80-F/pyrGAn-R and TtrpC-F/D-ku80-R, respectively . The promoter P gpdAt was amplified from the plasmid pXH102 using the primer pair PgpdAt-F743/PgpdAt-R .…”

Section: Methodsmentioning

confidence: 99%

“…To address these problems, an efficient marker-free gene-targeting system with high homologous recombination frequency and marker rescue capability was developed in A. terreus HZ01, as described previously. 24 The ku80-and pyrG-deficient mutant A. terreus HZ03 (A. terreus HZ01, Δku80::ptrA, ΔpyrG) was constructed and used as the parental strain for the deletion of lovF in this study.…”

Section: Acs Synthetic Biologymentioning

confidence: 99%

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Construction of an Efficient and Robust Aspergillus terreus Cell Factory for Monacolin J Production

Huang¹,

Tang

Zheng

et al. 2019

ACS Synth. Biol.

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Monacolin J is a key precursor for the synthesis of the cholesterol-lowering drug simvastatin. Industrially, monacolin J is manufactured through the alkaline hydrolysis of the fungal polyketide lovastatin, which is relatively complex and environmentally unfriendly. A cell factory for monacolin J production was created by heterologously introducing lovastatin hydrolase into Aspergillus terreus in our previous study. However, residual lovastatin remained a problem for the downstream product purification. In this study, we used combined metabolic engineering strategies to create a more efficient and robust monacolin J-producing cell factory that completely lacks lovastatin residue. The complete deletion of the key gene lovF blocked the biosynthesis of lovastatin and led to a large accumulation of monacolin J without any lovastatin residue. Additionally, the overexpression of the specific transcription factor lovE under the PgpdAt promoter further increased the titer of monacolin J by 52.5% to 5.5 g L–1. Interestingly, the fermentation robustness was also significantly improved by the expression of lovE. This improvement not only avoids the process of alkaline hydrolysis but also simplifies the downstream separation process.

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Biotechnological production of itaconic acid—things you have to know

Kuenz

Krull

2018

Appl Microbiol Biotechnol

108

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Itaconic acid is one of the basic chemicals for the polymer industry, which can be produced on the basis of renewable raw materials. Since the middle of the twentieth century, itaconic acid has been produced industrially using the filamentous fungus Aspergillus terreus. But the demand for the organic acid is low due to the high production costs compared to alternative petrochemical manufactured raw materials. The high production costs are based on a low final titer, low productivities, and the usage of pure sugars, purified molasses, or starch hydrolysates, since the fungus reacts very sensitively to impurities in a culture medium. This review provides a comprehensive overview of the most recent developments, including a spectrum of studied microorganisms and their capabilities for the production of itaconic acid. The technological achievements in the biotechnological production of itaconic acid are presented. Particular attention is paid to current achievements in terms of suitable alternative substrates and their applicability in fermentation processes. Also, the pathway of itaconic acid and especially the influences on the fermentation process, which must be known in order to achieve a high final titer of itaconic acid, a yield close to the theoretical yield, and high productivity.

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Establishing an efficient gene-targeting system in an itaconic-acid producing Aspergillus terreus strain

Abstract: This gene-targeting system is an efficient platform for sequential and multiple genetic modifications in A. terreus and is conducive to study biosynthesis mechanisms and to improve the production ability of itaconic acid and other products.

Cited by 18 publications

References 27 publications

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

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

Construction of an Efficient and Robust Aspergillus terreus Cell Factory for Monacolin J Production

Biotechnological production of itaconic acid—things you have to know

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