Metabolically Independent and Accurately AdjustableAspergillussp. Expression System

Abstract

Filamentous fungi are well-established expression hosts often used to produce extracellular proteins of use in the food and pharmaceutical industries. The expression systems presently used in Aspergillus species rely on either strong constitutive promoters, e.g., that for glyceraldehyde-3-phosphate dehydrogenase, or inducible systems derived from metabolic pathways, e.g., glaA (glucoamylase) or alc (alcohol dehydrogenase). We describe for Aspergillus nidulans and Aspergillus niger a novel expression system that utilizes the transcriptional activation of the human estrogen receptor by estrogenic substances. The system functions independently from metabolic signals and therefore can be used with low-cost, complex media. A combination of positive and negative regulatory elements in the promoter drives the expression of a reporter gene, yielding a linear dose response to the inducer. The off status is completely tight, yet the system responds within minutes to induction and reaches a level of expression of up to 15% of total cell protein after 8 h. Both Aspergillus species are very sensitive to estrogenic substances, and low-cost inducers function in the picomolar concentration range, at which estrogenic substances also can be found in the environment. Given this high sensitivity to estrogens, Aspergillus cells carrying estrogen-responsive units could be used to detect xenoestrogens in food or in the environment.In their natural environment, fungi use extracellular enzymes to gain access to complex, often water-insoluble carbon and nitrogen sources. Extracellular protein production by filamentous fungi usually is very efficient for homologous proteins, for which levels of grams per liter can be obtained. Aspergillus and Trichoderma strains have been reported to secrete up to 30 g of homologous proteins per liter in fermentation processes (e.g., glucoamylases) (11). The production of recombinant proteins of mammalian origin can be up to 4 orders of magnitude lower even when the same expression signals are used.The reason for this difference is not completely understood (2). Along with mRNA stability, codon usage, and translational efficiency, overloading the secretory machinery with a protein that has a "foreign" structure appears to be a critical bottleneck (16,34). High concentrations of such heterologous proteins can result in incorrect folding-leading to translational feedback inhibition by phosphorylation of the ␣ subunit of eukaryotic initiation factor 2 (23) and subsequent intracellular degradation of the misfolded protein. Thus, strong but highly regulatable expression systems are needed to control the flow of proteins through the secretory pathway.The use of strong promoters derived from housekeeping genes, such as those of the various fungal glyceraldehyde-3-phosphate dehydrogenase genes, has the disadvantage of continuous, growth-related expression levels (31). This problem may reduce yields of proteins susceptible to degradation or lead to cell death if the overexpressed foreign proteins are toxic to the ...

Sistema de expressão induzido por estradiol em <i>Saccharomyces cerevisiae</i>.

“…Entretanto, essas moléculas podem ser altamente tóxicas dependendo do microrganismo a ser utilizado como hospedeiro do sistema. Sendo assim, é de extrema importância a seleção e estudo de novos indutores (PACHLINGER et al, 2005).…”

“…Um modelo bastante interessante de indução para expressão de proteínas recombinantes é baseado em um tipo de promotor regulado pelo hormônio estrogênio (NALLEY; JOHNSTON; KODADEK, 2006;PACHLINGER et al, 2005). A regulação dirigida por esse tipo de indutor envolve a presença de um fator de transcrição quimérico constituído por um domínio de ligação ao DNA, um domínio de ativação e um domínio de ligação hormonal (QUINTERO et al, 2007).…”

“…Considerando que esse hormônio não é produzido em eucariotos inferiores, acredita-se que a utilização do hormônio não deve ter efeito sobre o genoma ou metabolismo do fungo (PACHLINGER et al, 2005).…”

“…Além do possível aumento da expressão da proteína de interesse, outra vantagem da utilização de estrogênio como indutor é o baixo custo dessas substâncias e a baixa ou nula interferência no metabolismo celular do organismo em estudo (PACHLINGER et al, 2005), criando uma vantagem adicional a esse tipo de sistema.…”

“…Pois suas propriedades (composição química, peso molecular, solubilidade, metabolismo, pH, entre outros) podem influenciar no metabolismo do microrganismo, sendo um aspecto importante a ser considerado (PACHLINGER et al, 2005). Portanto, é necessário identificar, desenhar e caracterizar novos sistemas de expressão, com potencial utilização industrial e mínimo ou nulo efeito no metabolismo do microrganismo.…”