Polycistronic Expression System for Pichia pastoris Composed of Chitino- and Chitosanolytic Enzymes

Michal B Kaczmarek; Katarzyna Struszczyk-Swita; Meng Xiao; Mirosława Szczęsna-Antczak; Tadeusz Antczak; Magdalena Gierszewska; Alexander Steinbüchel; Maurycy Daroch

doi:10.3389/fbioe.2021.710922

Polycistronic Expression System for Pichia pastoris Composed of Chitino- and Chitosanolytic Enzymes

Front Bioeng Biotechnol. 2021 Aug 18:9:710922. doi: 10.3389/fbioe.2021.710922. eCollection 2021.

Authors

Michal B Kaczmarek^{1

2}, Katarzyna Struszczyk-Swita², Meng Xiao¹, Mirosława Szczęsna-Antczak², Tadeusz Antczak², Magdalena Gierszewska³, Alexander Steinbüchel⁴, Maurycy Daroch¹

Affiliations

¹ School of Environment and Energy, Peking University Shenzhen Graduate School, Shenzhen, China.
² Institute of Molecular and Industrial Biotechnology, Lodz University of Technology, Lodz, Poland.
³ Department of Physical Chemistry and Physicochemistry of Polymers, Faculty of Chemistry, Nicolaus Copernicus University in Toruń, Toruń, Poland.
⁴ International Center for Research on Innovative Biobased Materials (ICRI-BioM), International Research Agenda, Lodz University of Technology, Lodz, Poland.

Abstract

Chitin is one of the most abundant biopolymers. Due to its recalcitrant nature and insolubility in accessible solvents, it is often considered waste and not a bioresource. The products of chitin modification such as chitosan and chitooligosaccharides are highly sought, but their preparation is a challenging process, typically performed with thermochemical methods that lack specificities and generate hazardous waste. Enzymatic treatment is a promising alternative to these methods, but the preparation of multiple biocatalysts is costly. In this manuscript, we biochemically characterised chitin deacetylases of Mucor circinelloides IBT-83 and utilised one of them for the construction of the first eukaryotic, polycistronic expression system employing self-processing 2A sequences. The three chitin-processing enzymes; chitin deacetylase of M. circinelloides IBT-83, chitinase from Thermomyces lanuginosus, and chitosanase from Aspergillus fumigatus were expressed under the control of the same promoter in methylotrophic yeast Pichia pastoris and characterised for their synergistic action towards their respective substrates.

Keywords: chitin; chitin deacetylase; chitinase; chitosan; chitosanase; enzymatic modification; polycistronic expression; self-processing 2A sequence.