A new method for screening and isolation of hypersecretion mutants in Aspergillus niger

Abstract

Although filamentous fungi have a unique property of secreting a large amount of homologous extracellular proteins, the use of filamentous fungi as hosts for the production of heterologous proteins is limited because of the low production levels that are generally reached. Here, we report a general screening method for the isolation of mutants with increased protein production levels. The screening method makes use of an Aspergillus niger strain that lacks the two major amylolytic enzymes, glucoamylase (GlaA) and acid amylase (AamA). The double-mutant strain grows poorly on starch and its growth is restored after reintroducing the catalytic part of the glucoamylase gene (GlaA512). We show that the fusion of a heterologous protein, a laccase from Pleurotus ostreatus (Pox2), to the catalytic part of glucoamylase (GlaA512-Pox2) severely hampers efficient production of the glucoamylase protein, resulting in a slow-growth phenotype on starch. Laccase-hypersecreting mutants were obtained by isolating mutants that displayed improved growth on starch plates. The mutant with the highest growth rate on starch displayed the highest laccase activity, indicating that increased glucoamylase protein levels are correlated with higher laccase production levels. In principle, our method can be applied to any low-produced heterologous protein that is secreted as a fusion with the glucoamylase protein.

The Aspergillus niger multicopper oxidase family: analysis and overexpression of laccase-like encoding genes

“…Although some of them have been homologous expressed in A. nidulans , being used as a reporter system [5], no overproduction studies have been described. In that sense, the performance of the GlaA signal sequence (ssGlaA), successfully used to improve the secretion of many proteins, including recombinant laccases [37-39], as well as different culture conditions, were analyzed in this work.…”

Physiological regulation of laccase and manganese peroxidase production by white-rot Basidiomycetes

“…The main issue delaying their implementation at industrial scale is the low yield of ligninolytic enzymes in most white-rot fungi. Although many recombinant organisms efficiently overproduce various industrial enzymes, high expression of laccase and peroxidases in heterologous systems has not been achieved yet and they still have to be obtained from natural sources (Weenink et al, 2006;Bailey et al, 2007). Moreover, ligninolytic enzymes of some basidiomycetes are formed in secondary metabolism; therefore, it 0168-1656/$ -see front matter © 2009 Elsevier B.V. All rights reserved.…”

Two Novel, Putatively Cell Wall-Associated and Glycosylphosphatidylinositol-Anchored α-Glucanotransferase Enzymes ofAspergillus niger

“…A. niger strain MGG029-⌬aamA (68) was used as a host for protein overexpression. This strain, derived from strain MGG029 (prtT glaA::fleo r pyrG), is deficient in the expression of several extracellular proteases, and it has no glucoamylase gene (glaA) and acid amylase gene (aamA), resulting in very poor growth on starch (68). Strain MA70.15 (pyrG Ϫ ku70::amdS) (46) was used for disruption of the agtA gene.…”