Secreted
Expression of Fungal Chitin Deacetylase into Yeast, Pichia pastoris.
Binesh Shrestha (1),
Willem F. Stevens (1), Françoise Le Hégarat (2)
(1) Bioprocess Technology/PT, Asian Institute of Technology, Thailand
(st018568@ait.ac.th); (2) Institut de Génétique et Microbiologie, Universite
Paris-Sud, France.
Pichia pastoris, a methylotrophic yeast, is a well known eukaryotic model for expression of eukaryotic as well as prokaryotic genes. The gene encoding chitin deacetylase from a fungus, Colletotrichum lindemuthianum UPS9 was cloned and expressed into Pichia pastoris. Chitin deacetylase is an enzyme that catalyzes the conversion of chitin (a non-biodegradable biopolymer, second abundant after cellulose) into chitosan (a high value biodegradable fine chemical having wide applications) which has drawn a significant attention for its potentials in replacing harsh thermochemical processes to the controlled conversion of chitin to chitosan. The chitin deacetylase gene was cloned downstream of a gene encoding alcohol oxidase (AOX1) promoter derived from wild strain of Pichia pastoris and also downstream of alpha-Factor signal sequence derived from Saccharomyces cerevisiae. Expression of the AOX1 gene is tightly regulated and induced by methanol to very high levels. Complete genomic DNA and cDNA from the source organism were cloned to observe the ability of Pichia pastoris for post-translational processing of the protein. Small scale and large scale expression studies were carried out in shake flask and in bioreactor to compare the expression level. The growth of the clone is highly favoured in aerated system of bioreactor. The clones were screened for its ability to act on native and partially deacetylated chitin substrate.