title: Improving the Secretory Expression of an -Galactosidase from Aspergillus niger in Pichia pastoris.
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.
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, and CAS Key Laboratory of Biofuels at Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, Shandong 266101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.|Sino-Danish Center for Education and Research, Beijing, 100190, China.
authors.forename: Xianliang
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, and CAS Key Laboratory of Biofuels at Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, Shandong 266101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Fang
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, and CAS Key Laboratory of Biofuels at Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, Shandong 266101, China.
authors.forename: Dongfei
authors.initials: D
authors.lastname: Han
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, and CAS Key Laboratory of Biofuels at Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, Shandong 266101, China.
authors.forename: Wenxia
authors.initials: W
authors.lastname: Yang
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, and CAS Key Laboratory of Biofuels at Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, Shandong 266101, China.
authors.forename: Feifei
authors.initials: F
authors.lastname: Qi
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, and CAS Key Laboratory of Biofuels at Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, Shandong 266101, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.affiliation: Shandong Provincial Key Laboratory of Synthetic Biology, and CAS Key Laboratory of Biofuels at Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao, Shandong 266101, China.
authors.forename: Shengying
authors.initials: S
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001234:Aspergillus niger / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D019149:Bioreactors; D003001:Cloning, Molecular; D003062:Codon; D005285:Fermentation; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007218:Industrial Microbiology; D000432:Methanol / Q000378:metabolism / Q000494:pharmacology; D010843:Pichia / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D043484:Proprotein Convertases / Q000737:chemistry; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D000519:alpha-Galactosidase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism
chemical_list: D003062:Codon; D005656:Fungal Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000519:alpha-Galactosidase; D043484:Proprotein Convertases; C058751:KEX2 protein, S cerevisiae; D000432:Methanol