Design principles for stable and balanced multigene expression in plant synthetic biology

Abstract Plant synthetic biology is increasingly moving from single-gene transformation toward multigene systems that reconstruct metabolic pathways, produce valuable proteins, implement synthetic circuits, and respond to environmental inputs. However, stable multigene expression in plants is limited by failures that arise across multiple biological layers, including DNA delivery and integration, chromatin context, transcription and RNA processing, translation, protein targeting, and physiological burden. In this review, we discuss multigene expression as a context-aware and burden-aware design problem rather than a simple DNA assembly problem. We first summarize major mechanisms that destabilize transgene expression, including structural rearrangement, transcriptional and post-transcriptional gene silencing, inefficient 3’-end processing, transcriptional interference, combinatorial part effects, and metabolic burden. We then describe part-level and architecture-level strategies for improving expression stability. Finally, we highlight the need for context-aware and burden-aware validation of every introduced gene across stable events, generations, tissues, and environmental conditions.