Production of the biotechnologically relevant AFP from Aspergillus giganteus in the yeast Pichia pastoris

Abstract

The mould Aspergillus giganteus produces a basic, low molecular weight protein (AFP) showing in vitro and in vivo antifungal properties against important plant pathogens. AFP is secreted as an inactive precursor containing an amino-terminal extension of six amino acids (If-AFP) which is later removed to produce the active protein. The molecular basis to explain this behavior and the features that determine the fungal specificity of this protein are not completely solved. In this work, the mature AFP (AFP*) and a version of AFP with an extended amino-terminal (proAFP) have been cloned and produced in the yeast Pichia pastoris. The two proteins have been purified to homogeneity and characterized from structural and functional points of view. AFP* produced is practically indistinguishable from the natural fungal protein in terms of its spectroscopic and antifungal properties while proAFP is mostly inactive under identical assay conditions. The availability of an active AFP protein produced in P. pastoris will allow getting further insights into the mode of action and targeting specificity of AFP by using site-directed mutagenesis approaches

The Epichloë festucae antifungal protein has activity against the plant pathogen Sclerotinia homoeocarpa, the causal agent of dollar spot disease

“…fischeri (NFAP) have been successfully expressed in P . pastoris 32, 34, 35 . Coding sequences of the long form (designated Efe - afpA -2) and the major mature form (designated Efe - afpA -3) of Efe -AfpA were cloned into the P .…”

“…However, when mature forms of Efe -AfpA, and the similar PAF, AFP, and NFAP proteins were expressed in P . pastoris , all lacking the pro-sequence, they could fold into active forms 32, 34, 35 . Apparently the eukaryotic cellular environment of P .…”

“…The Efe - afpA coding sequences were amplified by PCR with oligonucleotides that introduced a Xho 1 site and the Kex2 site at the 5′ ends and a Not 1 site at the 3′ end. The Ste13 cleavage sites were not included since the STE13 protease has been reported to inefficiently cleave the resulting protein 34, 62 . The oligonucleotides used for PCR amplification of the coding sequences are presented in Table 1.…”

Highly active promoters and native secretion signals for protein production during extremely low growth rates in Aspergillus niger

“…The activity of antifungal protein is restricted to moulds with no cytotoxic effects detected on yeast, bacteria, plant and mammalians cells [20], which enables a wide-range of conceivable applications. The heterologous production of AFP failed so far in E. coli (own unpublished data), yet was successful in P. pastoris with 2.5 mg/l of mature protein [21] and in A. niger but with only low titres (350 µg/l [12]). Aspergillus niger itself is very sensitive against AFP with a MIC (minimal inhibitory concentration) of ~1 µg/ml (complete inhibition of germination) and ~80 µg/ml for the fungicidal effect on hyphae [22].…”

“…While shake flask production is not suitable for industrial applications, these data suggest that further optimization of perfusion fermentation is warranted. Elsewhere, groups using P. pastoris for AFP production have achieved yields of 2.5 mg/l [21]. However, all heterologous expression attempts are low in comparison to the homologous production host A. giganteus (30–40 mg/l), indicating further optimisation of all current systems is required for commerical use of AFP.…”