Finding needles in haystacks: identification of novel conserved PETase enzymes in Streptomyces

Abstract

The rising use of plastic results in an appalling amount of waste which scatters into the environment affecting environmental, animal, and human health. One of these plastics is PET which is mainly used for bottles and textiles. In this research, we investigate the PET degrading ability of the IsPETase homolog ScLipA from Streptomyces coelicolor. Of 96 different Streptomyces strains screened, 18 % were able to degrade the model substrate BHET. Three different variants of lipase A, named ScLipA, S2LipA and S92LipA were identified and analyzed in detail. The lipA gene was deleted from S. coelicolor M145 using CRISPR/Cas9, resulting in reduced BHET degradation. LipA overexpression in the knock-out background significantly enhanced BHET degradation. All three enzymes were expressed in E. coli BL21 for protein purification and biochemical analysis, showing that enzymatic activity most likely resides in a dimeric form of the enzyme. The optimum pH and temperature were determined to be pH 7 and 25 {degrees}C for all three variants. Using these conditions, the activity on BHET and amorphous PET film was investigated. S2LipA efficiently degraded BHET and caused roughening and small indents on the surface of PET films, consistent with PET-degrading activity. The frequent occurrence of the S2LipA variant in Streptomyces suggests an environmental advantage towards the degradation of more hydrophobic substrates such as these polluting plastics in the environment.