<p>Biological upcycling is a promising strategy for handling end-of-life plastics. However, current practices, which predominantly focus on yielding fixed single outputs, face challenges in meeting diverse and evolving product demands. Here we present a programmable microbial assembly line that enables the efficient and versatile upcycling of plastic waste into various end products. The assembly line involves a deconstruction strain that catabolizes pretreated polyethylene terephthalate into pyruvate, serving as a universal building block, and a set of interchangeable production strains that transform this building block into tailorable outputs. Such a design allows a rapid, combinatorial assembly of microbial strains into diverse biomanufacturing systems. Accordingly, programmable upcycling is achieved, yielding successful production of a wide range of final products, including chemicals, fuels, enzymes, biopolymers and electricity. This work establishes a reconfigurable valorization platform, providing insights into biological treatment of plastic waste as well as into need-driven, sustainable biomanufacturing.</p>