Commercial Levels of Chymosin Production by Aspergillus

Dunn-Coleman, Nigel; Bloebaum, Peggy; Berka, Randy M.; Bodie, Elizabeth A.; Robinson, Nancy; Armstrong, Gale; Ward, Michael P.; Przetak, Melinda; Carter, Gina L.; LaCost, Regina; Wilson, Lori J.; Kodama, Katherine H.; Baliu, Enrique F.; Bower, B.; Lamsa, Michael; Heinsohn, Henry

doi:10.1038/nbt1091-976

Cited by 153 publications

(63 citation statements)

References 12 publications

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“…Fungi produce a wide spectrum of both intracellular and extracellular proteases many of which are active in the secretory pathway presenting major problems to heterologous protein production . Strains with lowered protease activity have been created by specific gene deletion or by mutagenesis (Mattern et al, 1992) and have been successfully used as improved production hosts (Contreras et al, 1991 ;Dunn-Coleman et al, 1991).…”

Section: Gene Fusionsmentioning

confidence: 99%

Regulation of secreted protein production by filamentous fungi: recent developments and perspectives

MacKenzie

Jeenes

Belshaw

et al. 1993

Journal of General Microbiology

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Section: Gene Fusionsmentioning

confidence: 99%

Regulation of secreted protein production by filamentous fungi: recent developments and perspectives

MacKenzie

Jeenes

Belshaw

et al. 1993

Journal of General Microbiology

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“…In general, A. niger is an excellent host for the production of fungal proteins but high concentrations of heterologous (non-fungal) products are notoriously difficult to obtain [2,3]. Though commercially interesting levels of recombinant bovine chymosin, an enzyme used for cheese manufacture, have already been reported in the early nineties [4,5], other proteins, for example, biopharmaceuticals such as interleukin-6 [3], tissue plasminogen activator [6], single-chain antibodies [7], and hepatitis B pseudoviral particles [8] are still just found in milligram per liter quantities in the culture medium. Unsatisfactory low yields are often caused by proteolytic degradation of the target protein [6,9], with improvements being obtained by modifying culture conditions [6,10,11] and developing strains with less proteases [12,13].…”

Section: Introductionmentioning

confidence: 99%

Agitation effects on morphology and protein productive fractions of filamentous and pelleted growth forms of recombinant Aspergillus niger

El‐Enshasy

Kleine

Rinas

2006

Process Biochemistry

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Recombinant Aspergillus niger genetically engineered to produce glucose oxidase using the constitutive gpdA promoter and the glucoamylase signal sequence for secretion was grown in batch cultures at agitation speeds of 200 -800 rpm covering the industrial relevant power input range of 0.1 -5 W kg -1 . The growth morphology ranged from large pellets with an average diameter of 1500 µm at low power input up to micropellets embedded in a filamentous network at high power input. A correlation of agitation intensity with growth morphology and glucose oxidase production revealed an increase of the protein production capability with the change of the growth morphology from pelleted to filamentous growth forms. However, the exposure to higher shear stress with increasing power input also resulted in lower biomass yields as well as increased transient formation of polyol (xylitol) and higher final concentrations of oxalic acid. The highest specific production rates were found in young filamentous growth forms at high power input. Although intermediate agitation intensity leading to small pellets became more favorable during prolonged cultivation. An acridine orange staining procedure discriminating between RNA rich (red) and RNA poor regions (green) of the fungal biomass proved that active protein production is restricted to filamentous growth forms and the outer layer of fungal pellets. A correlation between the RNA rich fraction of the biomass determined by image analysis and the productivity is shown.

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“…In addition, A. oryzae has the ability to secrete large amounts of proteins and has recently become a favorable host for recombinant protein production (5). By use of a series of classical random mutagenesis and screening procedures, hypersecretion mutants of heterologous proteins in aspergilli have been obtained (9,49). However, the secretion yields of heterologous proteins are low compared to those of homologous proteins or proteins from closely related fungal species and generally do not exceed tens of milligrams per liter (16).…”

mentioning

confidence: 99%

Codon Optimization Increases Steady-State mRNA Levels in Aspergillus oryzae Heterologous Gene Expression

Tokuoka

Tanaka

Ono

et al. 2008

Appl Environ Microbiol

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We investigated the effect of codon optimization on the expression levels of heterologous proteins in Aspergillus oryzae, using the mite allergen Der f 7 as a model protein. A codon-optimized Der f 7 gene was synthesized according to the frequency of codon usage in A. oryzae by recursive PCR. Both native and optimized Der f 7 genes were expressed under the control of a high-level-expression promoter with their own signal peptides or in a fusion construct with A. oryzae glucoamylase (GlaA). Codon optimization markedly increased protein and mRNA production levels in both nonfused and GlaA-fused Der f 7 constructs. For constructs with native codons, analysis by 3 rapid amplification of cDNA ends revealed that poly(A) tracts tended to be added within the coding region, producing aberrant mRNAs that lack a termination codon. Insertion of a termination codon between the carrier GlaA and native Der f 7 proteins in the GlaA fusion construct resulted in increases in mRNA and secreted-carrier-GlaA levels. These results suggested that mRNAs without a termination codon as a result of premature polyadenylation are degraded, possibly through the nonstop mRNA decay pathway. We suggest that codon optimization in A. oryzae results in elimination of cryptic polyadenylation signals in native Der f 7, thereby circumventing the production of truncated transcripts and resulting in an increase in steady-state mRNA levels.The filamentous fungus Aspergillus oryzae has been used in the production of fermented foods, such as sake, soy sauce, and miso (soybean paste), in Japan for over a thousand years. In addition, A. oryzae has the ability to secrete large amounts of proteins and has recently become a favorable host for recombinant protein production (5). By use of a series of classical random mutagenesis and screening procedures, hypersecretion mutants of heterologous proteins in aspergilli have been obtained (9, 49). However, the secretion yields of heterologous proteins are low compared to those of homologous proteins or proteins from closely related fungal species and generally do not exceed tens of milligrams per liter (16). In order to improve the expression levels of heterologous proteins, several trials have been conducted, and some strategies have been reported to be effective in increasing the level of heterologous protein production (39). These results provide information on how to increase the expression levels of heterologous genes; however, there is little information on the mechanisms hampering heterologous gene expression. In this study, we investigate the effect of codon optimization on heterologous gene expression. Such optimization has been effective in improving secretion levels of heterologous proteins in several hosts (19). Studies of heterologous gene expression through codon optimization have reported improved heterologous protein production in filamentous fungal species, such as Aspergillus awamori (2,15,35), Aspergillus niger (27, 35), Neurospora crassa (13, 35), and Trichoderma reesei (47). Based on studies of ...

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Commercial Levels of Chymosin Production by Aspergillus

Cited by 153 publications

References 12 publications

Regulation of secreted protein production by filamentous fungi: recent developments and perspectives

Regulation of secreted protein production by filamentous fungi: recent developments and perspectives

Agitation effects on morphology and protein productive fractions of filamentous and pelleted growth forms of recombinant Aspergillus niger

Codon Optimization Increases Steady-State mRNA Levels in Aspergillus oryzae Heterologous Gene Expression

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