Reduction of Extracellular Proteases Increased Activity and Stability of Heterologous Protein in $${ Aspergillus}$$ A s p e r g i l l u s $${ niger}$$ n i g e r

Kamaruddin, Nurhaida; Storms, Reginald; Mahadi, Nor Muhammad; Illias, Rosli Md.; Bakar, Farah Diba Abu; Murad, Abdul Munir Abdul

doi:10.1007/s13369-017-2914-3

Cited by 14 publications

(17 citation statements)

References 40 publications

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“…The construction of protease-deficient strains of Aspergillus, Trichoderma and Myceliophthora sp. for application in heterologous expression of polypeptides, including cellulolytic enzymes, has been described [6,[109][110][111]. Protease deficient strains have been obtained by mutagenesis and by direct gene knockout strategies [112].…”

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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Section: Recombinant Productionmentioning

confidence: 99%

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

Lübeck

2022

Microorganisms

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“…An alternative and more efficient approach is the use of protease-deficient strains [124]. Conventional mutagenesis and genetic engineering were applied to several Aspergillus species in order to disrupt genes that encode extracellular proteases, e.g., aspergillopepsin A (pepA) [125,126] or protease regulatory genes [126,127] (Table 10). The specific mutants exhibit reduced extracellular proteolytic activity and often appear to be more efficient producers of heterologous proteins than the wild types [124].…”

Section: Proteasesmentioning

confidence: 99%

“…Deletion of pepA in A. awamori resulted in an aspergillopepsin A-deficient mutant, with decreased proteolytic activity [125] and able to produce higher levels of bovine chymosin (~430 mg/L), when compared to the control strain (A. awamori strain GC12-∆argB3, ∆pyrG5:~180 mg/L) [128]. A. niger mutants lacking the transcription factor PrtT, which regulates expression of both aspergillopepsin A and B genes (pepA and pepB) [129], showed only 1-2% of the parental strain extracellular protease activity [126] and were used to produce highly stable heterologous cutinase with 1.7-fold increased activity [127]. Stability: Cutinase activity retained at 80% over the entire 14-day incubation period, while the parental lost more than 50% of their initial activities after six days of incubation and retained negligible activity after 14 days -Deletion of dppV and pepA in A. nidulans P. sanguineus laccase activity 500,000 U/mL compared to 40,000 U/mL in the control strain 12.5 [51] Deletion of mnn9 and pepA in A. nidulans P. sanguineus laccase activity 300,000 U/mL compared to 40,000 U/mL in the control strain 7.5 [51] Research on Aspergillus protease repertoire and development of molecular tools for multiple gene targeting allowed the disruption of multiple protease-related genes in a single production host, a successful tactic to further decrease proteolytic degradation and therefore improve protein production titers (Table 10) [51,118,130,131].…”

Section: Proteasesmentioning

confidence: 99%

“…Subsequently, A. oryzae quintuple and decuple protease gene disruptants produced even higher HLY amounts (26.5 mg/L and 35.8 mg/L, respectively) [130,131]. Although multiple protease-related gene disruptions appear to be a time-consuming and tedious procedure [127], it is a strategy commonly used for the optimization of heterologous protein production in several Aspergillus systems of industrial interest [99,132,133].…”

Section: Proteasesmentioning

confidence: 99%

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Aspergillus: A Powerful Protein Production Platform

et al. 2020

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Aspergilli have been widely used in the production of organic acids, enzymes, and secondary metabolites for almost a century. Today, several GRAS (generally recognized as safe) Aspergillus species hold a central role in the field of industrial biotechnology with multiple profitable applications. Since the 1990s, research has focused on the use of Aspergillus species in the development of cell factories for the production of recombinant proteins mainly due to their natively high secretion capacity. Advances in the Aspergillus-specific molecular toolkit and combination of several engineering strategies (e.g., protease-deficient strains and fusions to carrier proteins) resulted in strains able to generate high titers of recombinant fungal proteins. However, the production of non-fungal proteins appears to still be inefficient due to bottlenecks in fungal expression and secretion machinery. After a brief overview of the different heterologous expression systems currently available, this review focuses on the filamentous fungi belonging to the genus Aspergillus and their use in recombinant protein production. We describe key steps in protein synthesis and secretion that may limit production efficiency in Aspergillus systems and present genetic engineering approaches and bioprocessing strategies that have been adopted in order to improve recombinant protein titers and expand the potential of Aspergilli as competitive production platforms.

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“…At present, tannase, xylanase, mannase and asparaginase have been successfully expressed at a high level in strains with a lower endogenous protein background [13,14]. In another example, Kamaruddin et al [15] increased the yield of cutinase by 36-fold in A. niger by down-regulating the protease expression.…”

Section: Introductionmentioning

confidence: 99%

Improved Homologous Expression of the Acidic Lipase from Aspergillus niger

Si-Yuan¹,

Xu²,

Xiang³

2020

Journal of Microbiology and Biotechnology

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In this study, the acidic lipase from Aspergillus niger (ANL) was homologously expressed in A. niger. The expression of ANL was significantly improved by the expression of the native ANL with the introns, the addition of the Kozak sequence and the optimization of the signal sequences. When the cDNA sequence of ANL fused with the glaA signal was expressed under the gpdA promoter in A. niger, no lipase activity could be detected. We then tried to improve the expression by using the full-length ANL gene containing three introns, and the lipase activity in the supernatant reached 75.80 U/ml, probably as a result of a more stable mRNA structure. The expression was further improved to 100.60 U/ml by introducing a Kozak sequence around the start codon due to a higher translation efficiency. Finally, the effects of three signal sequences including the cbhI signal, the ANL signal and the glaA signal on the lipase expression were evaluated. The transformant with the cbhI signal showed the highest lipase activity (314.67 U/ml), which was 1.90-fold and 3.13-fold higher than those with the ANL signal and the glaA signal, respectively. The acidic lipase was characterized and its highest activity was detected at pH 3.0 and a temperature of 45ºC. These results provided promising strategies for the production of the acidic lipase from A. niger.

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Reduction of Extracellular Proteases Increased Activity and Stability of Heterologous Protein in $${ Aspergillus}$$ A s p e r g i l l u s $${ niger}$$ n i g e r

Cited by 14 publications

References 40 publications

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

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

Aspergillus: A Powerful Protein Production Platform

Improved Homologous Expression of the Acidic Lipase from Aspergillus niger

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