Seamless and orthogonal expression of genetic parts in polyhydroxyalkanoate (PHA)-producing bacterial chassis for plastic bio-upcycling applications.

Polyhydroxyalkanoate (PHA)-producing bacteria represent a powerful synthetic biology chassis for waste bioconversion and bio-upcycling where PHAs can be produced as the final products. In this study, we present a seamless plasmid construction for orthogonal expression of recombinant PET hydrolase (PETase) in model PHA-producing bacteria and . To this end, this study described seamless cloning and expression methods utilizing SureVector (SV) system for generating pSV-Ortho-PHA (pSVOP) expression platform in bioengineered and . Genetic parts specifically Trc promoter, pBBR1 origin of replication, anchoring proteins and signal sequences were utilized for the transformation of pSVOP-based plasmid in electrocompetent cells and orthogonal expression of PETase in both and . Validation steps in confirming functional expression of PETase activity in corresponding PETase-expressing strains were also described to demonstrate seamless and detailed methods in establishing bioengineered and as whole-cell biocatalysts tailored for plastic bio-upcycling.•Seamless plasmid construction for orthogonal expression in PHA-producing bacteria.•Step-by-step guide for high-efficiency generation of electrotransformants of and •Adaptable methods for rapid strain development (Design, Build, Test and Learn) for whole-cell biocatalysis.