Cloning and heterologous expression of the extracellular alpha-galactosidase from Aspergillus fumigatus in Aspergillus sojae under the control of gpdA promoter
Introduction
α-Galactosidase (alpha-d-galactoside galactohydrolase; EC 3.2.1.22) is an exoglycosidase that catalyzes the hydrolysis of the terminal non-reducing α-1.6-linked α-galactose residues from oligosaccharides such as melibiose, raffinose, and stachyose and from polymeric galactomannans and galactolipids. Some α-galactosidases can also catalyze transgalactosylation reactions, especially at high substrate concentration [1]. A. fumigatus α-galactosidase has also been shown to catalyze efficient transgalactosylation reaction to a variety of mono- and oligosaccharides, including the fragments of galactomannans main chain [2]. α-Galactosidases occur widely in microorganisms, plants and animals [3]. Microorganisms have the advantage of high production yields, and among them, fungal galactosidases are the most suitable for technological applications mainly due to their extracellular secretion, acidic pH optima, and broad stability profiles. Filamentous fungi have been extensively employed on an industrial scale for many decades in the production of a variety of enzymes [4].
α-Galactosidases have a number of biotechnological, medical and industrial applications. The most important industrial application is in the sugar-making industry [5] by the removal of raffinose from sugar beet molasses to facilitate the crystallization and improve the yield of sucrose.
The α-galactosidase gene of interest is located on chromosome 5 of A. fumigatus genome. During growth of the strain, A. fumigatus IMI 385708, on 2% locust bean gum (LBG), Puchart et al. [6] observed high levels of extracellular α-galactosidase production.
A. fumigatus is a saprophytic, thermotolerant, and haploid fungus. Among the 182 recognized species of Aspergillus [7], A. fumigatus is the most common human and animal pathogen. The opportunistic fungus mainly affects immunocompromised patients [8]. However, A. fumigatus is a good producer of many hydrolytic enzymes. The ability of A. fumigatus α-galactosidase to debranch polymeric legume seed galactomannans is quite rare among microbial α-galactosidases. This feature is common to α-galactosidases from Penicillium simplicissimum AGLI, P. ochrochloron and A. niger [9], [10], [11]. Degree of polymeric galactomannans degalactosylation by A. fumigatus enzyme was found to be the highest among microbial α-galactosidases and approached that by enzymes from plants [12], [13].
A fairly new expression host from A. sojae ATCC strain was developed by Margreet Heerikhuisen, Cees van den Hondel and Peter Punt, TNO, The Netherlands. A. sojae ATCC11906 (pyrG−) strain [14], having low proteolytic activity and being a uridine auxotrophic mutant which is unable to produce the enzyme orotidine-5-monophosphate decarboxylase, involved in the biosynthesis of uridine, was used for heterologous expression.
In the present study, α-galactosidase gene of the human pathogen A. fumigatus (aglB) was ligated onto the fungal expression vector, pAN52-4 (accession no. Z32699), and transformed into A. sojae ATCC11906 (pyrG−) strain, a GRAS organism.
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Strains, media, and cultivation
The thermotolerant filamentous fungus, A. fumigatus strain IMI 385708, which was formerly known as Thermomyces lanuginosus IMI 158749 (International Mycological Institute, Kew, Surrey, UK) [15] was cultivated on YpSs agar (4 g/L yeast extract, 1 g/L% K2HPO4, 0.5 g/L MgSO4·7H2O, 15 g/L starch, 2% agar) at 45 °C until sporulation and then stored at 4 °C. For induction of α-galactosidase gene, the culture was grown in YpSs broth using 0.5% LBG instead of starch at 45 °C and 155 rpm.
Escherichia coli
Results
According to the derived amino acid sequence of A. fumigatus, aglB gene encodes an extracellular protein of 447 amino acids with a 22 amino acids long signal peptide. The plasmid DNA of the construct was sequenced and aligned with the published sequence of A. fumigatus genomic DNA (data not shown). As described in Section 2.2, the mature protein coding region of aglB was cloned by PCR followed by a ligation on vector pAN52-4. On the construct after signal peptide cleavage 5 extra amino acids
Discussion
The expression system used in this study was very efficient and allowed us to obtain high heterologous expression of α-galactosidase in recombinant A. sojae in the absence of complex polymeric substrate. The use of strong promoters derived from housekeeping genes, such as the fungal glyceraldehyde-3-phosphate dehydrogenase gene, has the advantage of constitutive, growth-associated production of heterologous proteins. The 5 extra amino acids on the N-terminus of the transformant mature protein
Acknowledgements
We thank Dr. Punt from TNO, The Netherlands for A. sojae ATCC11906 (pyrG−) strain and pAN52-4 expression vector.
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