Improving the Secretory Expression of an - α-Galactosidase from Aspergillus niger in Pichia pastoris

Xianliang Zheng, Bo Fang, Dongfei Han, Wenxia Yang, Feifei Qi, Hui Chen, Shengying Li, Israel Silman

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Abstract

alpha-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 alpha-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 alpha-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 (Mut(s)) strain of AGA-I with the specific P1' site substitution (Glu to Ile) demonstrated remarkable extracellular a-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 alpha-galactosidase in P. pastoris, thus holding great potential for industrial application.
Original languageEnglish
Article numbere0161529
JournalP L o S One
Volume11
Issue number8
Number of pages12
ISSN1932-6203
DOIs
Publication statusPublished - 2016
Externally publishedYes

Keywords

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Aspergillus niger
  • Base Sequence
  • Bioreactors
  • Cloning, Molecular
  • Codon
  • Fermentation
  • Fungal Proteins
  • Gene Expression
  • Industrial Microbiology
  • Methanol
  • Pichia
  • Proprotein Convertases
  • Protein Sorting Signals
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • alpha-Galactosidase
  • KEX2 protein, S cerevisiae
  • Medicine (all)
  • Biochemistry, Genetics and Molecular Biology (all)
  • Agricultural and Biological Sciences (all)
  • MULTIDISCIPLINARY
  • HIGH-LEVEL EXPRESSION
  • HUMAN ALPHA-GALACTOSIDASE
  • ALCOHOL OXIDASE
  • SACCHAROMYCES-CEREVISIAE
  • THERMOMYCES-LANUGINOSUS
  • METHYLOTROPHIC YEAST
  • CLEAVAGE SITES
  • GENE
  • CLONING
  • PURIFICATION
  • Genetics - General
  • Genetics - Plant
  • Biochemistry studies - General
  • Enzymes - General and comparative studies: coenzymes
  • methanol
  • alpha-galactosidase
  • Fungi, Microorganisms, Nonvascular Plants, Plants
  • pharmaceutical industry
  • extracellular activity
  • microbial cell factory

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