Abstract
α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1’ residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Muts) strain of AGA-I with the specific P1’ site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application.
Highlights
As a subfamily of exo-glycosidases, α-galactosidases (D-galactoside galactohydrolase, EC. 3.2.1.22) are broadly distributed in microorganisms, plants and animals
To select an appropriate yeast strain for secretory expression of the α-galactosidase from A. niger (AGA), the recombinant plasmid pPICZαA-aga was transformed to P. pastoris X33 (Mut+, the methanol utilization plus phenotype) and P. pastoris KM71H (Muts, the methanol utilization slow phenotype), respectively
Through homologous recombination between the recombinant vector and the yeast genome, multiple chromosomal integration of the expression cassette consisting of the aga gene with its native signal peptide sequence replaced by the α-factor signal peptide sequence from S. cerevisiae, and a selectable zeocin resistance gene, onto the AOX1 locus of yeast genome were achieved
Summary
As a subfamily of exo-glycosidases, α-galactosidases (D-galactoside galactohydrolase, EC. 3.2.1.22) are broadly distributed in microorganisms, plants and animals. 3.2.1.22) are broadly distributed in microorganisms, plants and animals These enzymes primarily catalyze the removal of α-1,6-linked terminal non-reducing galactose residues from various α-galactosides [1], such as melibiose, raffinose, stachyose, and others [2]. They display transgalactosylation activity [3] and hemagglutination activity [4]. Some α-galactosidases are important animal feed additives since they are able to enhance the nutritive quality and energy efficiency of feed by hydrolyzing non-metabolizable sugars in corns and legumes [6], thereby alleviating flatulence, diarrhea and anorexia of animals The use of these enzymes in animal farming industries can benefit environments by lowering the PLOS ONE | DOI:10.1371/journal.pone.0161529. The use of these enzymes in animal farming industries can benefit environments by lowering the PLOS ONE | DOI:10.1371/journal.pone.0161529 August 22, 2016
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