Construction of a novel filamentous fungal protein expression system based on redesigning of regulatory elements

Zhang, Zhe; Xiang, Boyu; Zhao, Shengfang; Yang, Le; Yu, Chun-Hong; Hu, Yibo; Hu, Shengbiao

doi:10.1007/s00253-022-11761-0

Cited by 5 publications

(6 citation statements)

References 37 publications

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“…Thus, genetic modifications by knocking out the major extracellular proteins, such as glucoamylases, and the proteases, including PepA, PepB, PepD, and PepF, were performed in the A. niger natural strains [ 13 , 54 , 55 ]. However, conventional and laborious deletion of multiple genes in fungi is difficult using an inadequate number of transformants and selection markers [ 12 , 40 ]. Recently, a reasonable strategy of deletion of transcription factors to suppress the downstream extracellular protein expression is adopted to obtain a host with a lower background for enzyme production [ 9 , 40 ].…”

Section: Discussionmentioning

confidence: 99%

“…However, certain highly secreted background proteins, such as alpha-amylases and alpha-glucosidases, are also expressed with target enzyme under inducing conditions, which could restrict the ascension of target enzyme production [ 9 – 11 ]. Meanwhile, complex protein background is highly disadvantageous for the production of a specific protein or a relatively pure monocomponent enzyme, as it renders the purification of the target enzyme more tedious and costly [ 9 , 12 ].…”

Section: Introductionmentioning

confidence: 99%

“…Alternatively, Dong et al [ 15 ] knocked in two copies of trehalase genes from Myceliophthora thermophila at the loci of two highly expressed extracellular secreted alpha-amylases by the CRISPR/Cas9 tool, resulting in a 793.60% increase in trehalase activity along with a substantial abatement of protein background. Nonetheless, there is a high burden associated with achieving low secretion background hosts by multiple gene deletions, which necessitates the use of multiple screening markers in a single host and efficient gene targeting system [ 2 , 12 ]. Considering that the expression of amylolytic enzymes is controlled by the transcription activator AmyR, Zhang et al [ 9 ] constructed an host strain ∆ amyR with a low background of protein secretion by deleting amyR in A. niger CICC246.…”

Section: Introductionmentioning

confidence: 99%

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A novel sucrose-inducible expression system and its application for production of biomass-degrading enzymes in Aspergillus niger

Wang

Xie

Chang

et al. 2023

Biotechnol Biofuels

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Background Filamentous fungi are extensively exploited as important enzyme producers due to the superior secretory capability. However, the complexity of their secretomes greatly impairs the titer and purity of heterologous enzymes. Meanwhile, high-efficient evaluation and production of bulk enzymes, such as biomass-degrading enzymes, necessitate constructing powerful expression systems for bio-refinery applications. Results A novel sucrose-inducible expression system based on the host strain Aspergillus niger ATCC 20611 and the β-fructofuranosidase promoter (PfopA) was constructed. A. niger ATCC 20611 preferentially utilized sucrose for rapid growth and β-fructofuranosidase production. Its secretory background was relatively clean because β-fructofuranosidase, the key enzyme responsible for sucrose utilization, was essentially not secreted into the medium and the extracellular protease activity was low. Furthermore, the PfopA promoter showed a sucrose concentration-dependent induction pattern and was not subject to glucose repression. Moreover, the strength of PfopA was 7.68-fold higher than that of the commonly used glyceraldehyde-3-phosphate dehydrogenase promoter (PgpdA) with enhanced green fluorescence protein (EGFP) as a reporter. Thus, A. niger ATCC 20611 coupled with the PfopA promoter was used as an expression system to express a β-glucosidase gene (bgla) from A. niger C112, allowing the production of β-glucosidase at a titer of 17.84 U/mL. The crude β-glucosidase preparation could remarkably improve glucose yield in the saccharification of pretreated corncob residues when added to the cellulase mixture of Trichoderma reesei QM9414. The efficacy of this expression system was further demonstrated by co-expressing the T. reesei-derived chitinase Chi46 and β-N-acetylglucosaminidase Nag1 to obtain an efficient chitin-degrading enzyme cocktail, which could achieve the production of N-acetyl-D-glucosamine from colloidal chitin with a conversion ratio of 91.83%. Besides, the purity of the above-secreted biomass-degrading enzymes in the crude culture supernatant was over 86%. Conclusions This PfopA-driven expression system expands the genetic toolbox of A. niger and broadens the application field of the traditional fructo-oligosaccharides-producing strain A. niger ATCC 20611, advancing it to become a high-performing enzyme-producing cell factory. In particular, the sucrose-inducible expression system possessed the capacity to produce biomass-degrading enzymes at a high level and evade endogenous protein interference, providing a potential purification-free enzyme production platform for bio-refinery applications.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A novel sucrose-inducible expression system and its application for production of biomass-degrading enzymes in Aspergillus niger

Wang

Xie

Chang

et al. 2023

Biotechnol Biofuels

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“…The results are in agreement with those of PoGA15A from P. oxalicum GXU20 [ 6 ], demonstrating that the major RSDE Amy15A plays a dominant role in the degradation of raw starch. Unlike the heterologously expressed PoGA15A in Pichia pastoris , we obtained Amy15A by homologous expression in an engineered P. oxalicum host strain [ 29 ]. Although amino acid sequence alignment shows the high sequence identity between Amy15A and PoGA15A, some differences in enzymatic properties were found for the two amylolytic enzymes.…”

Section: Discussionmentioning

confidence: 99%

Genetic modifications of critical regulators provide new insights into regulation modes of raw-starch-digesting enzyme expression in Penicillium

Zhao

Xiang

Yang

et al. 2022

Biotechnol Biofuels

Self Cite

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Background Starch is a very abundant and renewable carbohydrate and an important feedstock for industrial applications. However, most starch-based products are not cost-efficient due to the high energy input needed in traditional enzymatic starch conversion processes. Raw-starch-digesting enzymes (RSDEs) from filamentous fungi have great commercial value in starch processing. However, the regulatory mechanisms associated with their production in filamentous fungi remain unknown. Results In this study, we reported the novel finding that cellulolytic fungus Penicillium oxalicum 114-2 has broad RSDE activity. Four regulators, including the amylase transcription activator AmyR, the catabolite repression repressor CreA, the group III G protein α subunit PGA3, and the nonhistone chromosomal protein HepA, have been found to play a crucial regulatory role in RSDE expression. Enzymatic assays revealed that RSDE production significantly increased after the overexpression of AmyR and HepA, the deletion of CreA and the dominant activation of PGA3. RT-qPCR analysis demonstrated that there is a mutual regulation mode between the four regulators, and then formed a cascade regulation mechanism that is involved in RSDE expression. Comparative transcriptomic analysis between the wild-type strain and genetically engineered strains revealed differentially expressed genes that may mediate the RSDE expression. Conclusions The four different types of regulators were systematically investigated and found to form a regulatory network controlling RSDE gene expression. Our results provide a new insight into the regulatory mechanism of fungal amylolytic enzyme expression and offer a theoretical basis to rationally improve the RSDE yield in the future.

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“…Pichia pastoris) and Saccharomyces cerevisiae) [94][95][96], the development of filamentous fungal expression platforms is much more complex, timeconsuming, and needs extensive development. Due to the great potentials and industrial interests, several reviews have addressed different aspects of recombinant production of proteins using filamentous fungi [3,26,93,[97][98][99].…”

Section: Recombinant Productionmentioning

confidence: 99%

Fungal Cell Factories for Efficient and Sustainable Production of Proteins and Peptides

Lübeck

2022

Microorganisms

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Filamentous fungi are a large and diverse taxonomically group of microorganisms found in all habitats worldwide. They grow as a network of cells called hyphae. Since filamentous fungi live in very diverse habitats, they produce different enzymes to degrade material for their living, for example hydrolytic enzymes to degrade various kinds of biomasses. Moreover, they produce defense proteins (antimicrobial peptides) and proteins for attaching surfaces (hydrophobins). Many of them are easy to cultivate in different known setups (submerged fermentation and solid-state fermentation) and their secretion of proteins and enzymes are often much larger than what is seen from yeast and bacteria. Therefore, filamentous fungi are in many industries the preferred production hosts of different proteins and enzymes. Edible fungi have traditionally been used as food, such as mushrooms or in fermented foods. New trends are to use edible fungi to produce myco-protein enriched foods. This review gives an overview of the different kinds of proteins, enzymes, and peptides produced by the most well-known fungi used as cell factories for different purposes and applications. Moreover, we describe some of the challenges that are important to consider when filamentous fungi are optimized as efficient cell factories.

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Construction of a novel filamentous fungal protein expression system based on redesigning of regulatory elements

Cited by 5 publications

References 37 publications

A novel sucrose-inducible expression system and its application for production of biomass-degrading enzymes in Aspergillus niger

A novel sucrose-inducible expression system and its application for production of biomass-degrading enzymes in Aspergillus niger

Genetic modifications of critical regulators provide new insights into regulation modes of raw-starch-digesting enzyme expression in Penicillium

Fungal Cell Factories for Efficient and Sustainable Production of Proteins and Peptides

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