Functional expression of engineered IsPETase and LCC variants in Drosophila melanogaster reveals host tolerance and differential catalytic activity

: Insects offer promising opportunities for organic waste bioconversion; however, they cannot efficiently degrade synthetic polymers such as polyethylene terephthalate (PET). Here, we generated transgenic Drosophila melanogaster lines to express in vitro-evolved variants of two PET-degrading enzymes with distinct biochemical properties: an engineered Ideonella sakaiensis PETase variant (TS-ΔIsPET) and a leaf-branch compost cutinase variant (TA-ΔLCC). Both enzymes, fused to a Drosophila gut-derived secretory signal, were produced and secreted by Drosophila cultured S2R+ cells and transgenic larvae. Upon secretion, TS-ΔIsPET and TA-ΔLCC enzymes were glycosylated, a post-translational modification that did not abolish their catalytic activity. Notably, TA-ΔLCC displayed ∼6-fold higher net esterase activity than TS-ΔIsPET in larval samples, and TA-ΔLCC-containing extracts depolymerised PET nanoparticles in vitro under enzyme-favourable conditions. Transgenic flies showed normal development, fertility and survival. Morphological and biochemical analyses confirmed that TA-ΔLCC expression did not alter midgut structure and function. Together, these results establish Drosophila melanogaster as a model for functional expression and comparative evaluation of engineered PET-degrading enzymes and identify TA-ΔLCC as a promising candidate for further evaluation in insect species relevant to plastic-contaminated waste bioconversion.