Abstract Description

Bioplastics, such as poly(lactic acid) (PLA), are considered sustainable alternatives to petrochemical derived polymers. However, more efficient waste processing, such as the use of recombinant enzymes for bioplastic hydrolysis, is required to improve the sustainability of bioplastics. Industrial strains of Saccharomyces cerevisiae are already used in large-scale fermentation and protein production platforms and thus represent a good choice for producing bioplastic hydrolysing enzymes. Often, however, methods to construct recombinant strains include the use of antibiotic resistance markers, which are undesirable for commercial applications. Use the acetamidase (amdSYM) recyclable dominant marker to develop a marker-less industrial Saccharomyces cerevisiae strain expressing CLE_PRD8, an engineered variant of the cutinase-like enzyme originating from Cryptococcus sp. S-2, which is capable of efficiently hydrolysing various bioplastic polymers and blends. The amdSYM marker and CLE_PRD8 gene were amplified to include appropriate homology and integrated into repeating delta sites in the genome of an industrial S. cerevisiae strain, namely Ethanol Red V1 (ER V1). Transformants were screened for bioplastic hydrolysing activity, and marker recycling was performed to remove the amdSYM marker from the best-performing transformants. Enzyme activity of the recombinant strains was evaluated using plate screening and quantitative turbidity-based enzyme activity assays, as well as SDS-PAGE analysis. The CLE_PRD8 gene was successfully expressed in the ER V1 S. cerevisiae strain. Hydrolysis of polycaprolactone (PCL) was observed in assays, and SDS-PAGE analysis of the crude supernatant confirmed extracellular production of the recombinant protein. Enzyme activity was significantly increased after excision of the amdSYM marker through counter-selection.