Construction of Heterologous Protein Expression System in Paecilomyces variotii using Gene Editing Technology
Hui-Gang Han and Hyeon-Su Ro*
Department of Bio&Medical Bigdata (BK21plus) and Research Institute of Life Sciences, Gyeongsang National University, Jinju, Korea
Paecilomyces variotii is a thermotolerant fungus belonging to the order Eurotiales that is widely found from food and soil. We isolated a strain of P. variotii (P.variotii MR1), surviving at the temperature of eukaryotic upper limit from a paper mill. P. variotii MR1 was under our scrutiny as a host in the production of useful proteins and its application at the elevated temperature. In this research, we firstly removed the spore-forming capability of it to avoid dissemination of spores to the ecosystem by using UV mutagenesis, yielded a conidia-deficient strain P. variotii UM7. An eGFP expression module was inserted to the chromosomal locus of UM7 disrupting pyrG through homologous recombination (HR) assisted by Cas9-gRNA ribonucleoprotein complex (RNP). PEG-mediated transformation with RNP and the eGFP expression module to the protoplast yielded 42 colonies surviving on 0.2% 5-FOA medium, as an indication of disruption of pyrG. Sequence analysis on the transformants revealed that 31% of them were HR products which carry the eGFP expression module. Meanwhile fragmented eGFP expression module was inserted to the target site through non-homologous end joining (NHEJ) with a substantial ration (67%). Deletion was relatively rare (2%) when it compared with other fungal gene editing. The expression of eGFP was confirmed at elevated temperatures. Our technology is under evaluation in the expression of thermostable protein.