Approaches for refining heterologous protein production in filamentous fungi

Fungi combine the advantages of a microbial system such as a simple fermentability with the capability of secreting proteins that are modified according to a general eukaryotic scheme. Filamentous fungi such as Aspergillus niger efficiently secrete genuine proteins but the secretion of recombinant proteins turned out be a difficult task. Aspergillus niger is an attractive organism because of its high secretion capacity and is frequently used as a model organism. Whereas high production yields can be obtained when homologous proteins are expressed, much lower amounts are obtained with the production of heterologous proteins. To fully exploit the potential of filamentous fungi, understanding of the molecular genetics, their physiology, and the glycosylation metabolism has to be investigated and clarified in more detail. This review summarizes recent developments in heterologous protein production by filamentous fungi and also generalizes the possibilities of improving the protein production by various genetic and bioprocessing approaches, thereby easing recognition of filamentous fungi as a relevant and reliable expression platform.     

Strategies for improving heterologous protein production from filamentous fungi

Despite the naturally high capacity for protein secretion by many species of filamentous fungi, secteted yields of many heterologous proteins have been comparatively low. The strategies for yield improvement have included the use of strong homologous promoters, increased gene copy number, gene fusions with a gene encoding a naturally well-secreted protein, protease-deficient host strains and screening for high yields following random mutagenesis. Such approaches have been effective with some target heterologous proteins but not others.Approaches used in heterologous protein production from filamentous fungi are discussed and a perspective on emerging strategies is presented.     

MAGGY,a retrotransposon in the genome of the rice blast fungusMagnaporthe grisea

Full-length copies of a previously described repetitive DNA sequence (CH2-8) were isolated from the genome of theMagnaporthe grisea strain 2539. One copy of the complete element was sequenced and found to resemble agypsy-like LTR retrotransposon. We named this element MAGGY (MAGnaporthe GYpsy-like element). MAGGY contains two internal ORFs putatively encoding Gag, Pol and Env-like proteins which are similar to peptides encoded by retroelements identified in other filamentous fungi. MAGGY was found to be widely distributed amongM. grisea isolates from geographically dispersed locations and different hosts. It was present in high copy number in the genomes of all nine rice-pathogenic isolates examined. By contrast,M. grisea strains isolated from other Gramineae were found to possess varying copy numbers of MAGGY and in some cases the element was completely absent. The wide distribution of MAGGY suggests that this element invaded the genome ofM. grisea prior to the evolution of rice-specific form(s). It may since have been horizontally transmitted to other sub-specific groups. One copy of MAGGY, corresponding to the element we sequenced, was located at identical locations in the genomes of geographically dispersed strains, suggesting that this copy of the element is a relatively ancient insertion.     

Natural products from filamentous fungi and production by heterologous expression

Applied Microbiology and Biotechnology - Filamentous fungi represent an incredibly rich and rather overlooked reservoir of natural products, which often show potent bioactivity and find...     

MAGGY, a retrotransposon in the genome of the rice blast fungusMagnaporthe grisea

Full-length copies of a previously described repetitive DNA sequence (CH2-8) were isolated from the genome of theMagnaporthe grisea strain 2539. One copy of the complete element was sequenced and found to resemble agypsy-like LTR retrotransposon. We named this element MAGGY (MAGnaporthe GYpsy-like element). MAGGY contains two internal ORFs putatively encoding Gag, Pol and Env-like proteins which are similar to peptides encoded by retroelements identified in other filamentous fungi. MAGGY was found to be widely distributed amongM. grisea isolates from geographically dispersed locations and different hosts. It was present in high copy number in the genomes of all nine rice-pathogenic isolates examined. By contrast,M. grisea strains isolated from other Gramineae were found to possess varying copy numbers of MAGGY and in some cases the element was completely absent. The wide distribution of MAGGY suggests that this element invaded the genome ofM. grisea prior to the evolution of rice-specific form(s). It may since have been horizontally transmitted to other sub-specific groups. One copy of MAGGY, corresponding to the element we sequenced, was located at identical locations in the genomes of geographically dispersed strains, suggesting that this copy of the element is a relatively ancient insertion.     

Absence of spermine in filamentous fungi.

Polyamines were examined in several yeasts and filamentous fungi. Whereas putrescine, spermidine, and spermine were present in the yeasts, spermine was not detected in any of the filamentous fungi.     

Autophagy in filamentous fungi

Autophagy is a ubiquitous, non-selective degradation process in eukaryotic cells that is conserved from yeast to man. Autophagy research has increased significantly in the last ten years, as autophagy has been connected with cancer, neurodegenerative disease and various human developmental processes. Autophagy also appears to play an important role in filamentous fungi, impacting growth, morphology and development. In this review, an autophagy model developed for the yeast Saccharomyces cerevisiae is used as an intellectual framework to discuss autophagy in filamentous fungi. Studies imply that, similar to yeast, fungal autophagy is characterized by the presence of autophagosomes and controlled by Tor kinase. In addition, fungal autophagy is apparently involved in protection against cell death and has significant effects on cellular growth and development. However, the only putative autophagy proteins characterized in filamentous fungi are Atg1 and Atg8. We discuss various strategies used to study and monitor fungal autophagy as well as the possible relationship between autophagy, physiology, and morphological development.     

丝状真菌的细胞凋亡

凋亡是一种程序性细胞死亡类型,为多细胞生物发育和维持生命所必需的,也普遍存在于细菌等原核生物和酵母、丝状真菌等真核生物中。丝状真菌既具有酵母和哺乳动物共有的凋亡同源蛋白,也具有酵母所不具备的哺乳动物凋亡同源蛋白,所以其凋亡机制较酵母更为复杂,而又较哺乳动物简单。凋亡在丝状真菌的发育、繁殖、衰老等过程中具有重要的作用。近年,丝状真菌作为新的凋亡研究的模式生物被广泛研究,而且进展迅速。综述丝状真菌的凋亡现象和检测方法,丝状真菌中凋亡的生物学功能,丝状真菌凋亡的诱导条件,以及丝状真菌凋亡相关基因的功能研究进展。     

The C-terminal chromodomain-like module in the integrase domain is crucial for high transposition efficiency of the retrotransposon MAGGY

MAGGY is a Ty3/Gypsy retrotransposon, which was identified in the rice blast fungus Magnaporthe oryzae. Some Ty3/Gypsy retrotransposons, including MAGGY, contain a chromodomain-like module (CLM) in the C-terminus of the integrase domain. We have made a series of MAGGY mutants to examine the role of the CLM in the transposition activity of the element. Introduction of a mutation at different positions in the MAGGY integrase revealed that a loss or alteration of the CLM resulted in a drastic decrease in the transposition activity of the element. Our results indicate that the CLM may confer high transposition activity to the element.     

Heterologous expression of genes in filamentous fungi.

Isolation of some biologically important proteins from natural sources was found to be too expensive or scarcely possible (human proteins). The problem could be solved by expression of heterologous genes. Many biologically active proteins have been successfully expressed in filamentous fungi, some of them, however, at a low level. Thus, improvement of this technique appears to be a very important task. The process comprises several steps. Some of them, such as efficient transformation, vector construction, processing of signal sequences, post-translational modifications and secretion of the expressed proteins, have been intensively investigated. This review presents obstacles and problems encountered in expression of heterologous genes and discusses strategies of development in this area.     

Proteomics of filamentous fungi

Proteomic analysis, defined here as the global assessment of cellular proteins expressed in a particular biological state, is a powerful tool that can provide a systematic understanding of events at the molecular level. Proteomic studies of filamentous fungi have only recently begun to appear in the literature, despite the prevalence of these organisms in the biotechnology industry, and their importance as both human and plant pathogens. Here, we review recent publications that have used a proteomic approach to develop a better understanding of filamentous fungi, highlighting sample preparation methods and whole-cell cytoplasmic proteomics, as well as subproteomics of cell envelope, mitochondrial and secreted proteins.     

Nucleases in the autolysis of filamentous fungi

RNase and DNase activities were studied in seven fungi of the subdivisions Ascomycotina, Zygomycotina and Basidiomycotina during their autolysis, and extracellular and intracellular RNase and DNase were found. RNase specific activity reached higher levels than DNase specific activity in the culture liquid and mycelial extract, except in Aspergillus nidulans. Generally maximal RNase specific activities were observed at the onset of autolysis in the culture liquid. In the mycelial extract an increase in this activity with the incubation time was observed, except in A. nidulans and Coriolus versicolor. The highest values of DNase specific activities were found at the third day of autolysis in A. nidulans culture liquid and at the thirtieth day of autolysis in Schizophyllum commune mycelial extract. A possible relationship between the culture liquid pH during the autolysis of the studied fungi and the levels of DNase specific activity was observed.     

Gene expression systems for filamentous fungi.

The extraordinary capacity of filamentous fungi to produce large quantities of extracellular protein, together with the advent of DNA-mediated fungal transformation, has resulted in rapid advances in the development of gene expression systems for filamentous fungi. This review focuses on recent developments in the expression of both fungal and non-fungal genes and improvements to the host.