| Abstract [eng] |
Biocatalysis is defined as the use of enzymes or whole cells to speed up chemical reactions. In order to meet the growing demand of biocatalysts in industry, it is crucial to have an effective gene expression system, that could be applied for a wide range of targeted enzymes. Since the problem of protein aggregation is still a very common issue in enzyme production, the focus of developing a prosperous expression systems is very relevant in academia and industry. The aim of this study was to develop a convenient expression system in yeast Kluyveromyces marxianus. Laccase (Lacc) from Bacillus pumilus and β-carbonic anhydrase (β-CA) from Bacilus mojavensis were selected as target proteins, that have never been expressed in K. marxianus. The genes of three proteins – maltose-binding protein (MBP), endopolygalacturonase (EPG) and triosephosphate isomerase (TPI) – were used as chaperones for increasing the solubility. Lacc cassettes (TPIprM-MBP-Lacc, TPIprE-EPG-Lacc, TPIprT-TPI-Lacc) and β-CA cassettes (TPIprM-MBP-CA, TPIprE-EPG-CA, TPIprT-TPI-CA) were assembled using overlapping DNA sequences between the fusing fragments. All of them were successfully cloned into pCAST cloning vector by homologous recombination in E. coli and Lacc cassettes were verified by sequencing. The three sequenced Lacc expression cassettes were further inserted into KLEF expression vector resulting in final DNA constructs for the expression of Lacc. K. marxianus were successfully transformed with the construct of KLEF-TPIprT-TPI-Lacc. |
